Pancreatic Cancer

Pancreatic Cancer

M. D. ANDERSON SOLID TUMOR ONCOLOGY SERIES Series Editor: Raphael E. Pollock, M.D., Ph.D. PUBLISHED VOLUMES Liver Cancer Edited by STEVEN A. CURLEY, M.D. Breast Cancer Edited by S. EVA SINGLETARY, M.D. Pancreatic Cancer Edited by DOUGLAS B. EVANS, M.D., PETER W.T. PISTERS, M.D., and JAMES L. ABBRUZZESE, M.D.

Andrew M. Lowy • Steven D. Leach Philip A. Philip Editors

Pancreatic Cancer

Editors Andrew M. Lowy University of California San Diego La Jolla, CA USA

Steven D. Leach Johns Hopkins University School of Medicine Baltimore, MD USA

Philip A. Philip Wayne State University Detroit, MI USA

Series Editor Raphael E. Pollock University of Texas M.D. Anderson Cancer Center Houston, TX USA

ISBN: 978-0-387-69250-0 e-ISBN: 978-0-387-69252-4 DOI: 10.1007/978-0-387-69252-4 Library of Congress Control Number: 2007934646 © 2008 Springer Science + Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Preface

This volume of the M.D. Anderson Solid Tumor Oncology Series details the significant developments in the field of pancreatic cancer research, diagnosis, and treatment that have occurred in the past 5 years. The authors represent a diverse group of experts who have endeavored to provide a historical perspective on pancreatic cancer care, combined with a practical evaluation of available data, to allow clinicians to assimilate these facts into their treatment algorithms. While progress on the clinical front has been slower than desired, the ability to perform large-scale clinical trials in both the adjuvant and advanced disease settings has improved dramatically. We have seen the development of the first mouse models that accurately recapitulate features of the human disease, tools that will enable pancreatic cancer researchers to ask new important questions aimed at early detection, biology, and therapy. An increased understanding of pancreatic cancer genetics and biology along with the rapid advancement of high throughput screening has led to the development of an unprecedented number of novel therapies for pancreatic cancer in preclinical and early phase clinical trials. The convergence of these events gives new hope that major advances in pancreatic cancer therapy will be realized during our lifetime. We greatly appreciate the exceptional contributions of the authors, each of which reflects their commitment to the field of pancreatic cancer research and treatment. Andrew M. Lowy, M.D. University of California, San Diego, Moores Cancer Center, La Jolla, California

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Contents

Section I

Molecular Pathology and Epidemiology

1 Spectrum of Human Pancreatic Neoplasia ........................................ N. Volkan Adsay, Duangpen Thirabanjasak, and Deniz Altinel

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2 Molecular Genetics of Pancreatic Cancer ............................................... Eric S. Calhoun and Scott E. Kern

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3 Pancreatic Intraepithelial Neoplasia .................................................... Ralph H. Hruban, Kieran Brune, Noriyoshi Fukushima, and Anirban Maitra

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4 Pathology and Molecular Biology of Intraductal Papillary Mucinous Neoplasms .................................................................... James G. Kench, Peter H. Cosman, Neil D. Merrett, and Andrew V. Biankin

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5 Familial Pancreatic Cancer .............................................................................. Kieran A. Brune and Alison P. Klein

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6 Hereditary Pancreatitis and Pancreatic Cancer ...................................... Julia B. Greer, Narcis O. Zarnescu, and David C. Whitcomb

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Section II

Pancreatic Cancer Biology

7 Pancreatic Progenitor Cells in Injury and Regeneration ...................... Solomon Afelik and Jan Jensen

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8 Development of the Endocrine and Exocrine Pancreas .................................................................................................................. 117 L. Charles Murtaugh, Jared Cassiano and Jean-Paul De La O 9 Models of Pancreatic Cancer: Understanding Disease Progression .................................................................................. Laleh G. Melstrom and Paul J. Grippo

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Contents

10 From Inception to Invasion: Modeling Pathways to Pancreatic Cancer................................................................................. Sunil R. Hingorani 11 Molecular Signaling Pathways in Pancreatic Cancer ........................ Genevieve M. Boland and Sarah P. Thayer 12 Pancreatic Cancer Genomics, Epigenomics, and Proteomics ....................................................................................... Michael Goggins

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Section III Pancreatic Cancer Staging 13 Imaging of Pancreatic Adenocarcinoma ............................................... Haydee Ojeda-Fournier and K. Ann Choe

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The Role of PET Scanning in Pancreatic Cancer ............................... M. Fernandez-Ulloa

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15 Endoscopic Ultrasound Staging .............................................................. William R. Brugge

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16 Laparoscopic Staging ......................................................................... Matthew H. Katz, Abdool R. Moossa, and Michael Bouvet

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Section IV

Surgery for Pancreatic Cancer

17 Pancreaticoduodenectomy: Past and Present ..................................... L. Andrew Shirley and Charles J. Yeo 18 Total Pancreatectomy for Treatment of Pancreatic Adenocarcinoma .................................................................................... Thomas Schnelldorfer Kaye M. Reid Lombardo, Michael B. Farnell, and Michael G. Sarr

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19 Distal Pancreatectomy ........................................................................... Andrew S. Resnick and Jeffrey A. Drebin

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20 Vascular Resection for Pancreatic Cancer ............................................ Waddah B. Al-Refaie and Jeffrey E. Lee

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Complications of Pancreaticoduodenectomy ........................................ Kyla Terhune, Nipun B. Merchant, and Alexander A. Parikh

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Controversies in the Surgical Management of Pancreatic Cancer................................................................................. Curtis J. Wray and Syed A. Ahmad

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Laparoscopic Pancreatic Resections .................................................... Attila Nakeeb

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Surgical Management of IPMN ........................................................... Stefano Crippa, Carlos Fernandez-del Castillo, and Andrew L. Warshaw

Section V

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Palliation of Pancreatic Cancer

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Cancer Pain and Symptom Management: Drug Therapy................. Jane Pruemer and Michelle Rockey

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Supportive Care: Cachexia, Anorexia Syndrome ............................... Michael John Tisdale

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Endoscopic Palliation ............................................................................ Nathan Schmulewitz

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Section VI 28

Multimodality Therapy of Pancreatic Cancer

State of the Art in Radiation Therapy for Pancreas Cancer.................. Aaron C. Spalding and Edgar Ben-Josef

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29 The Evolution of Chemoradiation Strategies for Locally Advanced Pancreatic Cancer ................................................................ A. William Blackstock and Stacy Wentworth

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Optimum Cytotoxic Therapy for Advanced Pancreatic Cancer ....... Sameer P. Desai and Mark M. Zalupski

31 Adjuvant Chemoradiation for Pancreatic Cancer: Past, Present, and Future ............................................................................... Michael C. Garofalo and William F. Regine 32 Neoadjuvant Treatment for Resectable and Locally Advanced Pancreatic Cancer ................................................................ Rebecca P. Petersen, Johanna C. Bendell, Brian G. Czito, and Douglas S. Tyler Section VII

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Emerging Targeted Therapies

33 An Overview of Clinical Trials of Targeted Therapies in Pancreatic Cancer............................................................................... Bassel El-Rayes 34 Utility of Animal Models in Pancreatic Cancer Research ................................................................................... Asfar S. Azmi, Mussop Mohammad, Ahmed O. Kaseb, Fazlul H. Sarkar, and Ramzi M. Mohammad 35 K-ras Inhibitors and Pancreatic Cancer ............................................. Steven R. Alberts

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36 HER Family of Receptors as Treatment Targets in Pancreatic Cancer................................................................................. Bhaumik B. Patel and Adhip P.N. Majumdar

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37 GSK-3β Inhibition in Pancreatic Cancer ............................................ George P. Kim and Daniel D. Billadeau

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38 Inactivation of NF-κB ............................................................................ Fazlul H. Sarkar and Yiwei Li

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39 Novel Targets for the Treatment of Pancreatic Cancer I: Insulin-like Growth Factor Receptor ................................................... Chris H. Takimoto

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40 Novel Targets for the Treatment of Pancreatic Cancer II: The Hedgehog Signaling Pathway........................................................ Chris H. Takimoto

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Development of Vaccine Therapy for Pancreas Cancer...................... Dung Le, Elizabeth M. Jaffee, and Dan Laheru

42 Novel Approaches in Chemoradiation for Localized Pancreas Cancer ..................................................................................... Christopher H. Crane 43 Incorporation of Genomics and Proteomics in Drug and Biomarker Development ................................................................ David G. Heidt, David Misek, David M. Lubman, and Diane M. Simeone

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44 Pitfalls in Clinical Trials and Future Directions ................................. Philip A. Philip, Lance K. Heilbrun, and Judith Abrams

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Index ................................................................................................................

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Contributors

Judith Abrams, PhD Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, USA N. Volkan Adsay, MD Emory University Hospitals, Department of Pathology, Atlanta, Georgia, USA Solomon Afelik, PhD Barbara Davis Center for Childhood Diabetes, University of Colorado, HSC, Denver, Colorado, USA Syed A. Ahmad, MD University of Cincinnati, Barrett Cancer Center, Cincinnati, Ohio, USA Steven R. Alberts, MD Mayo Clinic College of Medicine, Division of Medical Oncology, Rochester, Minnesota, USA Waddah B. Al-Refaie, MD The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA Deniz Altinel, MD Emory University Hospital, Atlanta, Georgia, USA Asfar S. Azmi, PhD Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA Johanna C. Bendell, MD Duke University Medical Center, Division of Medical Oncology, Department of Medicine, Durham, North Carolina, USA

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Contributors

Edgar Ben-Josef, MD University of Michigan, Department of Radiation Oncology, Ann Arbor, Michigan, USA Andrew V. Biankin, BMedSc, MB, BS, PhD Garvan Institute of Medical Research, and Bankstown Hospital, Sydney, NSW, Australia Daniel D. Billadeau, MD Mayo Clinic, Division of Oncology Research, Rochester, Minnesota, USA A. William Blackstock, MD Wake Forest University, Department of Radiation Oncology, Winston-Salem, North Carolina, USA Genevieve M. Boland, MD, PhD Massachusetts General Hospital, Department of Surgery, Boston, Massachusetts, USA Michael Bouvet, MD University of California, San Diego and Moores Cancer Center, La Jolla, California, USA William R. Brugge, MD Massachusetts General Hospital, Boston, Massachusetts, USA Kieran A. Brune, BS, The Sol Goldman Pancreatic Cancer Research Center and the John Hopkins Medical Institutions, Baltimore, Maryland, USA Eric S. Calhoun, PhD John Hopkins University School of Medicine, Baltimore, Maryland, USA Jared Cassiano, MS University of Utah, Department of Human Genetics, Salt Lake City, Utah, USA Carlos Fernandez-del Castillo, MD Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA Kyuran Ann Choe, MD University of Cincinnati College of Medicine, Cincinnati, Ohio, USA Peter H. Cosman, BA, MBBS, PhD, FRACS Garvan Institute of Medical Research, Sydney, and Bankstown Hospital, Bankstown, Sydney, Austria

Contributors

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Christopher H. Crane, MD The University of Texas M. D. Anderson Cancer Center, Radiation Oncology Department, Houston, Texas, USA Stefano Crippa, MD Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA Brian G. Czito, MD Duke University Medical Center, Department of Radiation Oncology, Durham, North Carolina, USA Jean-Paul De La O, BA University of Utah, Department of Human Genetics, Salt Lake City, Utah, USA Sameer P. Desai, MD University of Michigan, Division of Hematology-Oncology, Ann Arbor, Michigan, USA Jeffrey A. Drebin, MD, PhD University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA Bassel El-Rayes, MD Karmanos Cancer Center, Wayne State University, Detroit, Michigan, USA Michael B. Farnell, MD Mayo Clinic College of Surgery, Rochester, Minnesota, USA Mariano Fernandez, MD University of Cincinnati College of Medicine, Cincinnati, Ohio, USA Noriyoshi Fukushima, MD University of Tokyo, Tokyo, Japan Michael C. Garofalo, MD University of Maryland School of Medicine, Radiation Oncology Department, Baltimore, Maryland, USA Michael Goggins, MD The Sol Goldman Pancreatic Cancer Research Center and the John Hopkins Medical Institutions, Baltimore, Maryland, USA Julia B. Greer, MD, MPH University of Pittsburgh Medical Center, Division of Gastroenterology, Hepatology and Nutrition, Pittsburgh, Pennsylvania, USA

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Contributors

Paul J. Grippo, PhD Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA David G. Heidt, MD University of Michigan Medical School, Department of Surgery, Ann Arbor, Michigan, USA Lance Heilbrun, PhD Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, USA Sunil R. Hingorani, MD, PhD Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, Washington, USA Ralph H. Hruban, MD The Sol Goldman Pancreatic Cancer Research Center and the John Hopkins Medical Institutions, Baltimore, Maryland, USA Elizabeth M. Jaffee, MD The Sidney Kimmel Comprehensive Cancer Center, John Hopkins University School of Medicine, Baltimore, Maryland, USA Jan Jensen, PhD Barbara Davis Center for Childhood Diabetes, University of Colorado, HSC, Denver, Colorado, USA Ahmed O. Kaseb, MD The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA Matthew H. Katz, MD The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA James G. Kench, BSc (Med) Garvan Institute of Medical Research, Sydney, and Institute of Clinical Pathology and Medical Research, Westmead, Australia Scott E. Kern, MD The Sidney Kimmel Comprehensive Cancer Center, John Hopkins University School of Medicine, Baltimore, Maryland, USA George P. Kim, MD Mayo Clinic College of Medicine, Division of Hematology-Oncology, Jacksonville, Florida, USA

Contributors

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Alison Klein, PhD The Sidney Kimmel Comprehensive Cancer Center, John Hopkins University School of Medicine, Baltimore, Maryland, USA Dan Laheru, MD The Sidney Kimmel Comprehensive Cancer Center, John Hopkins University School of Medicine, Baltimore, Maryland, USA Dung Le, MD The Sidney Kimmel Comprehensive Cancer Center, John Hopkins University School of Medicine, Baltimore, Maryland, USA Steven D. Leach, MD John Hopkins University School of Medicine, Baltimore, Maryland, USA Jeffrey E. Lee, MD The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA Yiwei Li, PhD Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA Kaye M. Reid Lombardo, MD Mayo Clinic College of Medicine, Rochester, Minnesota, USA Andrew M. Lowy, MD University of California, San Diego and Moores Cancer Center, La Jolla, California, USA David M. Lubman, PhD University of Michigan Medical School, Departments of Surgery and Chemistry, Ann Arbor, Michigan, USA Anirban Maitra, MBBS The Sol Goldman Pancreatic Cancer Research Center and the John Hopkins Medical Institutions, Baltimore, Maryland, USA Adhip P.N. Majumdar, PhD, DSc John D. Dingell VA Medical Center, Detroit, Michigan, USA Laleh G. Melstrom, MD Northwestern University, Feinberg School of Medicine, Chicago, Illinois, USA Nipun B. Merchant, MD Vanderbilt University School of Medicine, Nashville, Tennessee, USA

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Contributors

Neil D. Merrett, MBBS Bankstown Hospital, Bankstown, Sydney, Australia David Misek, PhD University of Michigan Medical School, Department of Surgery, Ann Arbor, Michigan, USA Mussop Mohammad , BSc Wayne State University School of Medicine, Internal Medicine, Detroit, Michigan, USA Ramzi M. Mohammad, PhD Wayne State University School of Medicine, Internal Medicine, Detroit, Michigan, USA Abdool R. Moossa, MD Department of Surgery, University of California, San Diego, , San Diego, California, USA L. Charles Murtaugh, PhD University of Utah, Department of Human Genetics, Salt Lake City, Utah, USA Attila Nakeeb, MD Indiana University School of Medicine, Indianapolis, Indiana, USA Haydee Ojeda-Fournier, MD University of California, San Diego and Moores Cancer Center, La Jolla, California, USA Alexander A. Parikh, MD Vanderbilt University School of Medicine, Nashville, Tennessee, USA Bhaumik B. Patel, MD John D. Dingell VA Medical Center, Detroit, Michigan, USA Rebecca P. Petersen, MD, MSc, Duke University Medical Center, Department of Surgery, Durham, North Carolina, USA Philip A. Philip, MD, PhD Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, USA Jane Pruemer, Pharm.D, BCOP James L. Winkle College of Pharmacy, University of Cincinnati, Barrett Cancer Center, Cincinnati, Ohio, USA

Contributors

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William F. Regine, MD University of Maryland School of Medicine, Department of Radiation Oncology, Baltimore, Maryland, USA Andrew S. Resnick, MD University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA Michelle Rockey, PhD, BCOP University Hospital, Cincinnati, Ohio, USA Fazlul H. Sarkar, PhD Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA Michael G. Sarr, MD Mayo Clinic College of Medicine, Rochester, Minnesota, USA Nathan Schmulewitz, MD University of Cincinnati, Division of Gastroenterology, Cincinnati, Ohio, USA Thomas Schnelldorfer, MD Mayo Clinic College of Medicine, Rochester, Minnesota, USA L. Andrew Shirley, MD Thomas Jefferson University, Philadelphia, Pennsylvania, USA Diane M. Simeone, MD University of Michigan Medical School, Departments of Surgery and Molecular and Integrative Physiology, Ann Arbor, Michigan, USA Aaron C. Spalding, MD, PhD University of Michigan, Department of Radiation Oncology, Ann Arbor, Michigan, USA Chris H. Takimoto, MD, PhD University of Texas Health Science Center at San Antonio, South Texas Accelerated Research Therapeutics, San Antonio, Texas, USA Kyla Terhuns, MD Vanderbilt University School of Medicine, Nashville, Tennessee, USA Sarah P. Thayer, MD, PhD Massachusetts General Hospital, Department of Surgery, Boston, Massachusetts, USA

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Contributors

Duangpen Thirabanjasak, MD Chulalongkorn University, Bangkok, Thailand Michael John Tisdale, PhD, DSc School of Life and Health Sciences, Aston University, Birmingham, United Kingdom Douglas S. Tyler, MD Duke University Medical Center, Department of Surgery, Durham, North Carolina, USA Andrew L. Warshaw, MD Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA Stacy Wentworth, MD Wake Forest University, Department of Radiation Oncology, Winston-Salem, North Carolina, USA David C. Whitcomb, MD, PhD University of Pittsburgh, Departments of Medicine, Cell Biology & Physiology, and Human Genetics; University of Pittsburgh Medical Center: University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA Curtis J. Wray, MD The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA Charles J. Yeo, MD Thomas Jefferson University, Philadelphia, Pennsylvania, USA Mark M. Zalupski, MD University of Michigan, Division of Hematology-Oncology, Ann Arbor, Michigan, USA Narcis O. Zarnescu, MD University of Pittsburgh Medical Center, Department of Surgery, Pittsburgh, Pennsylvania, USA

Section I

Molecular Pathology and Epidemiology

Chapter 1

Spectrum of Human Pancreatic Neoplasia N. Volkan Adsay, Duangpen Thirabanjasak, and Deniz Altinel

1 Introduction Classification of pancreatic neoplasia is based on their cellular lineage, i.e which histologic cell type of the pancreas they recapitulate, which is in essence the main determinant of their clinicopathologic and biologic characteristics. Virtually every cell type that exists in the pancreas has been described to have one or more neoplastic counterparts. Cellular components of the pancreas can be regarded under six categories (1) (Fig. 1.1): (1) acinar and (2) ductal, which together constitute the exocrine component of the organ responsible for the production of digestive enzymes (trypsin, chymotrypsin, lipases, and others) and their transportation to the duodenum, respectively; (3) islets of Langerhans, the endocrine component, which is responsible for the production of hormones involved in regulation of glucose and other metabolic activities; (4) ambiguous cells, such as centroacinar cells, which are less well characterized; (5) supportive elements, which encompass fibrous tissue, vessels, nerves, and other connective tissue elements; and (6) “potential” cells, such as stem cells, the existence of which is evidenced based on in vitro studies, but their representatives in normal human pancreata have not yet been clearly identified in histologic sections (2). In the ensuing text, an overview of the clinicopathologic characteristics of pancreatic neoplasia is discussed based on their lineage. Emphasis is given to those that are more common and not covered in the other chapters of this book.

2 Ductal Neoplasms The ducts, which give rise to the vast majority pancreatic neoplasms, in fact constitute the least sophisticated component of this organ (3). They are merely responsible for transportation of acinar products to the ampulla of Vater. It may not be surprising, however, that ducts are more prone to neoplastic transformation, considering that most adult cancers, such as breast, prostate, lung, and colon, arise from A.M. Lowy et al. (eds.), Pancreatic Cancer. doi: 10.1007/978-0-387-69252-4, © Springer Science + Business Media, LLC 2008

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Fig. 1.1 Normal pancreatic tissue. The majority of the epithelial elements in the pancreas are acinar cells, which drain their products into the terminal portion of the ductal system (the centroacinar cells) and then into increasingly larger ducts. In a normal pancreas, the ducts are lined by a single layer of cuboidal epithelial cells without obvious mucin. The third epithelial component, the endocrine elements, are represented as scattered nested clusters of cells within the islets of Langerhans

epithelia with high regenerative capacity and from cell types that have secretory properties and produce mucin-type glycoproteins. Moreover, the ducts are the only component of the pancreas exposed to the outside world (mutagens), and the fact that most pancreatic adenocarcinomas arise in the head of the organ may be related to this phenomenon. It is also important for cancer researchers to recognize that although ducts give rise to most common neoplasia of the pancreas, they constitute only a very small minority of pancreatic tissue (1) (see Fig. 1.1), and thus comparison of “normal” and “malignant” pancreas is not a comparison of non-neoplastic versus cancerous ducts, but rather normal acini versus malignant ducts, which is essentially like comparing apples and oranges. The ductal system begins with the centroacinar unit (see Fig. 1.1), and through intralobar and interlobar ducts, adjoins the main pancreatic duct, eventually opening into the duodenum. Ductal cells produce some amount of mucin, more so in larger ducts, presumably for protection and providing the appropriate milieu for the acinar enzymes. Findings that characterize ductal differentiation in the pancreas include tubular structures (lumen-forming units; also referred to as glands or ducts), cysts (columnar epithelial-lined cavities), papillae (finger-like projections from an epitheliallined surface), and mucin formation (which can be intracytoplasmic, luminal, or

1 Spectrum of Human Pancreatic Neoplasia

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stromal). Mucin-related glycoproteins and oncoproteins such as MUC1, CA19-9, CEA, and DUPAN are typically detectable by immunohistochemistry in mucinous ductal tumors. Expression of certain subsets of cytokeratin, such as CK19, and mutations in K-ras oncogene or loss of SMAD4/DPC4 are also fairly specific, and are typically lacking in endocrine or acinar tumors with a few exceptions. Rare scattered endocrine cells can be seen in almost any ductal tumor; however, evidence of acinar differentiation such as enzyme activity is exceedingly uncommon.

2.1

Invasive Ductal Adenocarcinoma

Invasive ductal carcinoma, also called pancreatobiliary type adenocarcinoma because of its morphologic and biologic kinship to biliary carcinomas (3), is the most common neoplasm of the pancreas, constituting >85% of pancreatic tumors that come to clinical attention. It is one of the deadliest of all cancers, with a 5-year survival 10% of pancreatic resections (29). An estimated 2% of invasive pancreatic carcinomas arise in association with these tumors. They are potentially curable tumors, but there are controversies regarding their management. It is important to recognize the clinicopathologic characteristics of this group and the differential features of the entities under this category. These tumors are reviewed in detail in other chapters of this book.

Fig. 1.10 Intraductal papillary mucinous neoplasia (IPMN) is characterized by florid arborizing growth of neoplastic ductal epithelium in papillary architecture

1 Spectrum of Human Pancreatic Neoplasia

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Serous Ductal Neoplasia

Serous adenomas (30–32) (Fig. 1.11) are the only pancreatic tumors of ductal lineage that lack conventional features of mucinous differentiation, possibly recapitulating centroacinar cells. Serous tumors can be considered as the innocent among ductal neoplasia, because unlike their mucinous kindreds they do not show any association with malignancy. They form relatively large masses (mean size, 9 cm; some up to 25 cm), and occur predominantly in elderly women (mean age 63; female/male = 3/1). They tend to be well demarcated. Typically, they are composed of thousands of small cysts, each measuring only millimeters, hence the synonym microcystic adenoma. However, rare “macrocystic” and “solid” examples of serous adenomas are also on record. Microcystic serous cystadenomas often have a central stellate scar. The defining microscopic feature is a simple, nonmucinous, cuboidal epithelium that contains intracytoplasmic glycogen, resulting in characteristic clear cytoplasm. Along the same lines, the cyst contents are depleted of the mucin-related glycoproteins and oncoproteins that are typically found in mucinous pancreatic tumors, a feature that may help in the preoperative diagnosis (33). Microscopically, these lesions are very similar to the cysts seen in Von-Hippel Lindau disease, and in fact some serous cystadenomas do show VHL gene alterations. Of interest, serous cystadenomas are often reported to coexist or “collide” with other pancreatic neoplasms and with congenital pathologic conditions (32). Malignant serous tumors (serous cystadenocarcinomas) (34–36) are exceedingly uncommon, and only the presence of metastases or angioinvasive growth separates

Fig. 1.11 Serous cystic adenoma is characterized by numerous minute cysts and glands that show low cuboidal appearance with clear cytoplasm, intermingling with the proliferated small capillaries

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these tumors from their benign counterparts (i.e., the cytological appearance is entirely bland). For practical purposes, serous cystadenomas limited to the pancreas are regarded as uniformly benign (37).

3 Endocrine Neoplasia Focal endocrine differentiation, especially in the form of scattered cells, is quite common in pancreatic neoplasia of ductal and acinar nature and is of no known biologic significance. It is also mostly disregarded in classification of tumors. The true endocrine tumors of the pancreas are the pancreatic endocrine neoplasms.

3.1

Pancreatic Endocrine Neoplasms

The vast majority of endocrine tumors of the pancreas are well differentiated pancreatic endocrine neoplasms (PENs, previously referred to as islet cell tumors) (13, 38–41). Grossly, PENs are usually solid, circumscribed, and fleshy, although multinodular and sclerotic examples occur. Rarely, cystic degeneration may occur (42– 44), with a central unilocular cyst lined by a cuff of viable tumor. PENs recapitulate the morphologic features of the islet cells by forming nested, gyriform, trabecular, or rarely also acinar or glandular patterns. The cells have characteristic endocrine features including round, monotonous nuclei with a salt-and-pepper chromatin pattern and moderate amounts of cytoplasm (Fig. 1.12). Almost half of PENs are clinically functional and present with signs or symptoms of inappropriate production of one or more hormones; these functional PENs are named based upon the hormonal syndrome (insulinoma, glucagonoma, gastrinoma, somatostatinoma, VIPoma, and others). Nonfunctional PENs (45, 46) constitute an ever-enlarging proportion of pancreatic endocrine tumors, as they are being detected more commonly as incidental findings on abdominal imaging studies. Most insulinomas follow a benign clinical course, likely because insulinomas are typically highly symptomatic and are detected and removed when they are very small. PENs associated with MEN-1 or other syndromes tend to be multifocal and less aggressive (47). In addition to grossly evident and usually functional PENs, MEN1 patients also have numerous endocrine microadenomas, defined as PENs 3 cm, functional PEN other than insulinoma, extrapancreatic or vascular invasion, high mitotic activity, high proliferation index (based on immunohistochemical staining for Ki-67), CK19 expression, and tumor aneuploidy; however, some PENs lacking all of these features may still metastasize. The most recent WHO classification (50, 51) separates “well-differentiated pancreatic endocrine tumors” (those lacking metastases) from “well-differentiated pancreatic endocrine carcinomas” (those with metastases) and further predicts “benign” or “malignant” behavior in the former group based on size, mitotic rate, and vascular invasion. Another recent proposal (52) divides PENs into low- and intermediate-grade groups based on the mitotic rate (less than or greater than 2 mitoses per 50 high power fields) and necrosis. The two groups exhibit a highly significant difference in prognosis, although no subset is specifically regarded to be utterly benign.

4 Acinar Neoplasia Acinar differentiation can sometimes be seen as a focal component of endocrine neoplasia. It is also a major component of pancreatoblastoma (see the following). Tumors with predominant or pure acinar differentiation are acinar cell carcinomas. No benign counterpart of these tumors has been clearly documented.

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Acinar Cell Carcinoma

Acinar cell carcinomas (ACCs) (53–56) of the pancreas are uncommon, accounting for no more than 1–2% of pancreatic carcinomas (3). Most patients are adults in the seventh decade, and there is a male predominance. Pediatric cases have also been described, but are very rare. The presenting symptoms are generally nonspecific. Elevated serum AFP levels are noted in some patients. A minority of patients present with a lipase (enzyme) hypersecretion syndrome characterized by subcutaneous fat necrosis, polyarthralgia, and occasionally eosinophilia. Most cases have liver metastases early in the course of the disease. The tumors are generally highly cellular, lacking the desmoplastic fibrous stroma of ductal adenocarcinomas (Fig. 1.13). The cellular population is monotonous and arranged in solid sheets and nests punctuated by acinar and small glandular spaces. The cytoplasm is moderate to focally abundant and shows eosinophilic granularity in the apical regions, reflecting aggregates of zymogen granules that are highlighted by the immunohistochemical stains for specific enzymes (trypsin, lipase, and chymotrypsin), of which trypsin is the most useful. Mutation in K-ras gene (seen in >90% of ductal carcinomas) is absent in ACCs. B-catenin is expressed. Rarely, ACCs may show intraductal growth or papillary/papillocystic patterns and mimic intraductal neoplasia (19). Some studies suggest a more indolent behavior for these variants. Rare ACCs presenting as cystic lesions are also on record.

Fig. 1.13 Acinar cell carcinoma. At high power, diffuse proliferation of cells having moderate amounts of amphophilic cytoplasm and basally located nuclei. Prominent nucleoli are also a characteristic feature

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Recently, a rare benign cystic lesion of acinar origin (acinar cell cystadenoma; cystic acinar transformation) (57), which is typically an incidental microscopic change, but also rarely may form a clinically detectable mass, has been described.

5 Neoplasms with Multiple Lineage (Mixed Carcinoma and Pancreatoblastoma) While aberrant differentiation seldom occurs in ductal neoplasia, it is fairly common in nonductal tumors, in particular acinar neoplasia often has a mixed component, usually endocrine, detectable by immunohistochemistry (55). If the latter constitutes >25% of the tumor, it is classified as mixed acinar–endocrine carcinoma, a tumor that appears to be biologically similar to pure acinar cell carcinoma (56, 58, 59). Mixed ductal–endocrine tumors and mixed ductal–acinar tumors are exceedingly rare. Those we have seen had the two components almost in a “collision” and behaved like a ductal carcinoma. Perhaps the ultimate example of a pancreatic tumor with “polyphenotypic” differentiation is pancreatoblastoma (Fig. 1.14). It is an extremely rare childhood tumor of the pancreas (mean age 4, with a second small peak in thirties) (60–64). Pancreatoblastomas are usually large (7–18 cm). Some cases are

Fig. 1.14 Pancreatoblastoma. Several squamoid corpuscles illustrated here that are composed of spindle to epithelioid cells with abundant cytoplasm and less dense nuclei are characteristics of pancreatoblastoma

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associated with elevated serum alpha-fetoprotein levels, and occasional cases are seen in association with Beckwith-Wiedemann (65, 66) or familial adenomatous polyposis coli syndromes (18). Pancreatoblastomas are malignant tumors, with a 5-year survival of about 25%, although children diagnosed before the development of metastases may be curable. Pancreatoblastomas are predominantly acinar neoplasms. Microscopically they have sheets of primitive appearing epithelial cells and acinar formations. A characteristic and peculiar microscopic finding is the squamoid nest, which is a small morular arrangement of squamoid cells (67), specific for this tumor type. Typically pancreatoblastomas exhibit all three lines of pancreatic differentiation, acinar, endocrine and ductal, with acinar elements being the most consistently present (62). Molecular, genetic findings of pancreatoblastomas are different than those of ductal adenocarcinoma (68).

6 Tumors of Uncertain Histogenesis 6.1

Solid-Pseudopapillary Tumor

Solid-pseudopapillary tumor (SPT) is a peculiar neoplasm of unknown origin (69, 70), and its obscure nature is reflected in the various descriptive names assigned to this tumor in the past, including papillary cystic tumor, solid and papillary tumor, solid and cystic tumor, Frantz tumor, and many others (71–74). It is seen predominantly in young women (mean age 25, >80% female) (72, 75–77). It is an indolent malignant neoplasm for which complete resection is curative in most cases. In a small percentage (about 15%), however, metastases are noted, usually either to liver or peritoneum. Metastases are typically present at the time of diagnosis. Even patients with metastases appear to have a protracted clinical course; fatality due to this tumor is exceedingly uncommon. A few highly aggressive SPTs have been reported that appear to exhibit high-grade malignant transformation of a conventional SPT (77). SPTs are relatively large tumors, with a mean size of 8–10 cm. Although they are typically solid cellular tumors, cystic degeneration is common. Degenerative changes combined with the preservation of the cells around the microvasculature create the pseudopapillary microscopic pattern. The cells are round to oval and often show nuclear grooves. Intracytoplasmic hyaline globules and aggregates of foamy macrophages are characteristic findings (Fig. 1.15). Calcifications may be seen. Overall the histologic picture may closely resemble that of endocrine neoplasia. The immunophenotype of this tumor is quite distinctive and interesting, but fails to disclose the line of differentiation of the cells. The tumor typically expresses vimentin, progesterone receptors, CD56, and CD10 and, to a lesser degree, keratin and synaptophysin (72, 75, 78). Chromogranin is negative, which is important for the differential diagnosis with PENs. Recently, β-catenin11 and cyclinD1 expression have been found in these tumors, suggesting an alteration in the wnt signaling pathway.

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Fig. 1.15 Solid-pseudopapillary tumor. The solid, cellular regions of this tumor surrounding the small vessels, form the extensive pseudopapillary pattern with the cleft-like areas in between those structures

7 Miscellaneous Cystic Pancreatic Lesions In addition to pseudocysts, cystic and intraductal mucinous neoplasms and serous cystic tumors, there are other uncommon cystic lesions in the pancreas (76, 79, 80). Lymphoepithelial cysts (LECs) (81) are usually peripancreatic rather than intrapancreatic, and they occur predominantly in adult men (mean age 52, M/F = 3/1). Unlike their salivary gland counterparts, pancreatic LECs do not show associations with autoimmune syndromes, HIV, or malignancies such as lymphoma. They may occur in any part of the pancreas, and may be unilocular or multilocular. They are characterized by variably keratinized squamous lined cysts immediately surrounded by a rim of lymphoid tissue, some with lymphoid follicles and a capsule. The cyst contents may extravasate into the cyst wall and cause an inflammatory reaction, including granulomas. LEC-like epidermoid cystic tumors may evolve in intrapancreatic accessory spleens. Dermoid cysts (81) are similar to LECs but lack the lymphoid tissue and show skin adnexal elements, including sebaceous glands. Lymphangiomas (82) are seen in young women (mean age 29, M/F = 1/3) and form endothelial-lined cysts surrounded by a rim of lymphoid tissue. Squamoid cyst of pancreatic ducts (Fig. 1.16) is a recently described entity that is probably reactive in nature but may produce high CEA levels (80). Congenital cysts and intestinal duplications may also form cystic lesions in the

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Fig. 1.16 Squamoid cyst of pancreas. Benign cystic lesion with notably high CEA level. The cysts can be either uniloculated or multiloculated, lined by squamous metaplastic epithelial cells usually containing concentrated concretion within the lumen

vicinity of the pancreas and periampullary region. These may have variable lining including respiratory type, intestinal, squamous, or transitional.

8 Pseudotumors As discussed in the ductal adenocarcinoma section, benign chronic inflammatory and fibrosing conditions of the pancreas may be difficult to distinguish from carcinomas, both clinically and pathologically. Chronic pancreatitis of any etiology, including alcoholic, obstructive, or even those with granulomatous inflammation such as mycobacterial infection, may lead to segmental fibrosis and a tumorous mass (8, 83). There are, however, certain subtypes of chronic pancreatitis that are especially prone to form pseudotumors and mimic carcinomas, as discussed in the following. Lymphoplasmacytic sclerosing pancreatitis (LPSP; also referred to as autoimmune or duct-centric pancreatitis) (11, 83–85) is often misdiagnosed as carcinoma. LPSP is typically seen in patients in the fourth to sixth decades. High serum IgG4 levels are very helpful in the preoperative diagnosis of LPSP. LPSP may be associated with other autoimmune disorders such as Sjögren’s syndrome and ulcerative colitis as well as with components of the multifocal fibrosclerosis syndrome (retroperitoneal and mediastinal fibrosis, Riedel’s thyroiditis, inflammatory pseudotumor of the orbit, and sclerosing cholangitis). Microscopically, dense periductal lympho-

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plasmacytic infiltrates, interstitial fibrosis with abundant myofibroblasts arranged in a storiform pattern, and obliterative venulitis are characteristic. Some forms of anatomic pancreatitis (i.e., pancreatic fibroinflammatory masses associated with anatomic or functional variations of the ducts and periampullary structures) also commonly form pseudotumors. Some of these appear to be centered around the minor papilla. Affected patients are predominantly men in their forties with a history of alcohol abuse. In some cases, the fibroinflammatory process is in the region between common bile duct, duodenum, and pancreas (so-called groove pancreatitis) (86). In others cystic change in the duodenal wall is prominent (paraduodenal wall cyst or cystic dystrophy of the duodenum) (86–88). Microscopically, these lesions have a dense myofibroblastic proliferation within which lobules of pancreatic tissue are scattered, with cystic ducts containing inspissated enzymatic secretions. Extravasated ductal contents may elicit a cellular stromal reaction as well as inflammation. These cases are referred to as paraduodenal pancreatitis (10). Some developmental abnormalities may also lead to pseudotumors. Recently, a solid and cystic hamartoma and a cellular hamartoma (89) have been reported to present as a pancreatic tumors in adults. Lipomatous hypertrophy (66) of the pancreas may also lead to a mass that can be mistaken as carcinoma. Adenomyomatous hyperplasia (90) of the ampulla, a common finding in the general population reported in 40% of autopsies, has also been implicated as the cause of biliary obstruction mimicking a periampullary carcinoma.

9 Mesenchymal Tumors Primary mesenchymal tumors of the pancreas are exceedingly uncommon (91, 92); however, those from neighboring sites may secondarily involve the pancreas. Especially gastrointestinal stromal tumors and retroperitoneal sarcomas may appear to be centered in the pancreas. A variety of benign mesenchymal tumors, including fibromatosis (desmoid tumor), solitary fibrous tumor, schwannoma, and several others have been reported in the pancreas. Primary sarcomas include primitive neuroectodermal tumor, synovial sarcoma, desmoplastic small round cell tumor (93), leiomyosarcoma, and malignant fibrous histiocytoma, all of which are largely documented in single case reports.

10 Secondary Tumors It is not too uncommon for a widely metastatic malignant neoplasm to involve the pancreas; however, the majority of these are clinically undetected lesions identified only at autopsy (3, 94–96). Autopsy studies have shown that most secondary

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Fig. 1.17 Metastatic renal cell carcinoma in pancreas. Note the infiltrative tumor cells with clear cytoplasm, intermixing with the surrounding benign pancreatic tissue.

tumors involving the pancreas are of pulmonary origin, followed by gastrointestinal carcinomas. There are, however, a few metastatic tumors that are prone to involve the pancreas in the absence of other metastatic foci, mimicking a primary carcinoma. Lymphomas and renal cell carcinomas (Fig. 1.17) appear to be the most common tumor types responsible for such cases. Renal cell carcinomas in particular are known to form polypoid ampullary nodules, or even to grow within the ducts.

References 1. Klimstra DS. Pancreas. In: Sternberg SS (ed.) Histology for pathologists. New York, Lippincott-Raven, 1997. 2. Vyas P AA, Sarkar F, Leach S, et al. In search of stem cell in pancreatic ducts: the nature of round, non-ductal appearing cells in human pancreatic ducts. Mod Pathol 2003, 16(1):287A. 3. Klimstra DS, Adsay N. Benign and malignant tumors of the pancreas. In: Odze R, Goldblum JR, Crawford JM (eds.) Surgical pathology of the GI tract, liver, biliary tract and pancreas. Philadelphia, Saunders, 2004. 4. Jemal A, Siegel R, Ward E, et al. Cancer statistics, CA Cancer J Clin 2006, 56:106–130. 5. Pavlidis N, Briasoulis E, Hainsworth J, et al. Diagnostic and therapeutic management of cancer of an unknown primary. Eur J Cancer 2003, 39:1990–2005. 6. Evans DB, Willet CG. Cancer of the pancreas. In: DeVita VT, Rosenberg SA (eds.) Cancer principle and practice of oncology. Philadelphia, Lippincott Williams & Wilkins, 2001, 1126–1161. 7. Adsay NV, Bandyopadhyay S, Basturk O, et al. Chronic pancreatitis or pancreatic ductal adenocarcinoma? Semin Diagn Pathol 2004, 21:268–276.

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50. Kloppel GHR, Longnecker DS, Adler G, et al. Tumours of exocrine pancreas. In: Hsa LA (ed.) World Health Organization classification of tumours: pathology and genetis tumours of the digestive system. Lyon, France, IARC Press 2000, pp 219–251. 51. Solcia E, Capella C, Klöppel G (eds.) Tumors of the pancreas. Washington, DC, American Registry of Pathology, 1997, pp 145–209. 52. Hochwald SN, Zee S, Conlon KC, et al. Prognostic factors in pancreatic endocrine neoplasms: an analysis of 136 cases with a proposal for low-grade and intermediate-grade groups. J Clin Oncol 2002, 20:2633–2642. 53. Holen KD, Klimstra DS, Hummer A, et al. Clinical characteristics and outcomes from an institutional series of acinar cell carcinoma of the pancreas and related tumors. J Clin Oncol 2002, 20:4673–4678. 54. Hoorens A, Lemoine NR, McLellan E, et al. Pancreatic acinar cell carcinoma. An analysis of cell lineage markers, p53 expression, and Ki-ras mutation. Am J Pathol 1993, 143:685–698. 55. Klimstra DS, Heffess CS, Oertel JE, et al. Acinar cell carcinoma of the pancreas. A clinicopathologic study of 28 cases. Am J Surg Pathol 1992, 16:815–837. 56. Ohike N, Kosmahl M, Kloppel G. Mixed acinar-endocrine carcinoma of the pancreas. A clinicopathological study and comparison with acinar-cell carcinoma. Virchows Arch 2004, 445:231–235. 57. Zamboni G, Terris B, Scarpa A, et al. Acinar cell cystadenoma of the pancreas: a new entity? Am J Surg Pathol 2002, 26:698–704. 58. Klimstra DS, Rosai J, Heffess CS. Mixed acinar-endocrine carcinomas of the pancreas. Am J Surg Pathol 1994, 18:765–778. 59. Ulich T, Cheng L, Lewin KJ. Acinar-endocrine cell tumor of the pancreas. Report of a pancreatic tumor containing both zymogen and neuroendocrine granules. Cancer 1982, 50:2099–2105. 60. Dhebri AR, Connor S, Campbell F, et al. Diagnosis, treatment and outcome of pancreatoblastoma. Pancreatology 2004, 4:441–451; discussion 52–53. 61. Horie A, Haratake J, Jimi A, et al. Pancreatoblastoma in Japan, with differential diagnosis from papillary cystic tumor (ductuloacinar adenoma) of the pancreas. Acta Pathol Jpn 1987, 37:47–63. 62. Klimstra DS, Wenig BM, Adair CF, et al. Pancreatoblastoma. A clinicopathologic study and review of the literature. Am J Surg Pathol 1995, 19:1371–1389. 63. Palosaari D, Clayton F, Seaman J. Pancreatoblastoma in an adult. Arch Pathol Lab Med 1986, 110:650–652. 64. Shorter NA, Glick RD, Klimstra DS, et al. Malignant pancreatic tumors in childhood and adolescence: The Memorial Sloan-Kettering experience, 1967 to present. J Pediatr Surg 2002, 37:887–892. 65. Drut R, Jones MC. Congenital pancreatoblastoma in Beckwith-Wiedemann syndrome: an emerging association. Pediatr Pathol 1988, 8:331–339. 66. Kerr N, Fukuzawa R, Reeve AE, et al. Beckwith-Wiedemann syndrome, pancreatoblastoma, and the wnt signaling pathway. Am J Pathol 2002, 160(4):1541–1542. 67. Nishimata S, Kato K, Tanaka M, et al. Expression pattern of keratin subclasses in pancreatoblastoma with special emphasis on squamoid corpuscles. Pathol Int 2005, 55:297–302. 68. Cao D, Maitra A, Saavedra JA, et al. Expression of novel markers of pancreatic ductal adenocarcinoma in pancreatic nonductal neoplasms: additional evidence of different genetic pathways. Mod Pathol 2005, 18:752–761. 69. Kissane JM. Carcinoma of the exocrine pancreas: pathologic aspects. J Surg Oncol 1975, 7:167–174. 70. Kissane JM. Pancreatoblastoma and solid and cystic papillary tumor: two tumors related to pancreatic ontogeny. Semin Diagn Pathol 1994, 11:152–164. 71. Kloppel G, Morohoshi T, John HD, et al. Solid and cystic acinar cell tumour of the pancreas. A tumour in young women with favourable prognosis. Virchows Arch A Pathol Anat Histol 1981, 392:171–183. 72. Kosmahl M, Seada LS, Janig U, et al. Solid-pseudopapillary tumor of the pancreas: its origin revisited. Virchows Arch 2000, 436:473–480.

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Chapter 2

Molecular Genetics of Pancreatic Cancer Eric S. Calhoun and Scott E. Kern

1 Introduction In cancer, genome stability is compromised. Ductal adenocarcinoma of the pancreas is no exception. Considerable progress in the identification and characterization of somatic and/or germline genetic alterations has provided the mechanistic foundations of this genetically complex disease. Numerous alterations, including chromosomal copy-number gains, amplifications and homozygous deletions, loss of heterozygosity (LOH) with and without copy number reduction, and balanced and unbalanced structural re-arrangements, are commonly observed (1–4). Gross chromosomal changes are often complemented by smaller, more subtle alterations affecting the open reading frames of proto-oncogenes, tumor suppressors, and genome caretaker genes (5–8). Continued evaluation of these and additional yet-unidentified genetic alterations should translate into rational diagnostic and treatment strategies. This chapter describes the spectrum and frequency of genetic alterations observed in ductal adenocarcinomas of the pancreas, as organized by the underlying presence of two, largely mutually exclusive, types of genome instability: chromosomal instability (CIN) and microsatellite instability (MIN). This distinction is justified, as each tumor type exhibits unique histologic and molecular characteristics (9).

2 Chromosomal Instability The vast majority of pancreatic cancers (97%) have CIN, characterized by numerous chromosomal copy-number gains and losses, amplifications and homozygous deletions, translocations, and inversions. At first glance, these alterations appear random. Careful examination of overlapping genomic alterations has, however, revealed the recurrent targeting of select genes for disruption. The spectrum of altered genes, as well as the types of alterations, makes each pancreatic tumor rather distinctive.

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Required Genetic Mutations

Disruptions of at least two genes in pancreatic cancer are nearly universal. The first, K-ras (12p12.1), is an oncogene. A GTP-binding and hydrolyzing enzyme, the K-ras protein functions as a second messenger in growth factor receptor signaling pathways that stimulate the transition through the G1 phase of the cell division cycle. This activity is dependent upon bound GTP, remaining active as long as the nucleotide remains unhydrolyzed. Somatic mutations affecting the GTP-binding pocket can disrupt the ability of K-ras to hydrolyze GTP, rendering the protein constitutively active. Mutations of the codons Gly12, Gly13, or Gln61 are commonly associated with constitutively active K-ras, although recurrent alterations in Ala146 in colorectal cancers suggest additional hotspots may exist (10, 11). Ninety-five percent of pancreatic cancers harbor activating mutations in K-ras, representing the highest fraction of any tumor type (12, 13). The COSMIC (Catalogue of Somatic Mutations In Cancer) database lists the most common alteration in pancreatic cancer as the Gly12Asp substitution, accounting for ∼50% of all pancreatic cancer cases, whereas total alterations in Gly12 account for ∼99% of all mutations (14). The paucity of Gly13 mutations is unusual in that significantly higher frequencies are noted in other cancers (e.g., colorectal cancer: Gly13Asp, 15.8%; total Gly13 mutations, 17.3%). Conversely, Gly12Arg substitutions are commonly observed in pancreatic cancer (11.6%), whereas they occur infrequently in colorectal cases (1.0%). The underlying reasons for these tumor-specific mutational preferences are unknown. The second (virtually required) disruption in pancreatic cancer involves CDKN2A (p16, 9p21.3), one of the most frequently altered genes in cancer. A classical tumor-suppressor, p16 regulates cell cycle progression by inhibiting cyclinDCDK4/6, the kinase complex responsible for initiating the G1/S phase transition. Inherited alterations in p16 cause FAMMM (familial atypical multiple mole melanoma), a syndrome characterized by numerous dysplastic nevi having atypical shape, color and/or size, a propensity for developing melanoma and a distinct risk of pancreatic cancer (nearly a 20-fold increased risk) (15–18). The incidence of p16 alterations in sporadic pancreatic cancer is dramatic, with inactivation of this gene occurring in 98% of cases. Unlike K-ras activation, however, gene disruption (i.e., inactivation) occurs through multiple mechanisms including nearly half by homozygous deletion, more than a third by intragenic mutations with LOH, and the remainder by epigenetic promoter silencing (19, 20).

2.2

Frequently Mutated Genes

Genes mutated at a frequency >25% suggest they provide a supportive (i.e., selective advantage) role in the development of cancer. The implication that they are not required, however, suggests that other mutations might occasionally substitute for

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the more common alterations in these genes. Three examples of genes in this category include SMAD4 (18q21.1), TP53 (p53, 17p13.1), and NCOA3 (AIB1, 20q12), and for each there is limited evidence of substitute gene targets (21, 22). Inactivation of SMAD4 occurs in approximately 45% (30% by homozygous deletion) of pancreatic cancers, but less frequently in other cancer types (23–26). Functional characterization has shown that the SMAD4 tumor-suppressor protein is TGFβ-responsive, translocating to the nucleus (with Smads 2 and 3) after TGFβ or activin receptor stimulation to activate expression of genes mediating growth inhibition. Missense mutations in this gene impair protein stability, among other functional defects (27–29). Another frequently mutated gene in pancreatic cancer, and one of the most frequently altered genes in all cancer types, is TP53 (p53, 17p13.1). The tumor-suppressive properties of p53 lie in its ability to transcriptionally activate target genes in response to cellular stresses such as DNA damage. TP53 blocks G1/S and G2/M transitions by increasing p21WAF1/CIP1 and SFN (14-3-3 sigma) expression, and induces programmed cell death in experimental settings (30–32). Inactivating mutations occur in 70% of pancreatic tumors, with most affecting the ability of the protein to bind to DNA and activate gene transcription (33, 34). Unlike other tumor suppressors, however, homozygous deletions affecting p53 occur very rarely; most mutations are missense mutations. The reason for this remains unknown, but the gene dense region at 17p13.1 may inhibit the formation of homozygous deletions as they would provide a selective disadvantage to cells experiencing the loss of multiple genes. NCOA3 (AIB1, 20q12) enhances the transcriptional activity of a number of steroid nuclear receptors and is the rate limiting step in estrogen-mediated growth signaling. High-level amplification of this gene has been reported in ∼10% of breast cancer tissues and four of nine pancreatic cancer cell lines (35, 36). In archival tissues, copy-number gains were observed in >37% of pancreatic cancers (37). The observance of such high frequencies of AIB1 gains and amplifications in pancreatic cancer was surprising as pancreatic ductal tissues are not normally controlled by endocrine stimulation. Some of these authors suggest that such an amplification “may indicate that estrogen receptor mediated transcriptional activation confers a growth advantage” to these cells. The clinical relevance of these findings remain to be determined, however, as pancreatic cancers have not yet been shown to have hormone dependence.

2.3

Low-Frequency Mutations

Despite very little variation in clinical outcome, pancreatic tumors have heterogeneous mutational backgrounds that give each tumor a unique molecular signature. Each genetic mutation, occurring in eosinophilic cytoplasm, or severe/ enlarged oval and hyperchroHigh-grade matic nuclei with pseudostratification. This type may show low papillae consisting of cells with amphophilic cytoplasm.

–

+

+

Pancreatobiliary

Thin branching complex papillae, Severe/ moderate eosinophilic High-grade cytoplasm, enlarged hyperchromatic nuclei.

+

–

+

Oncocytic

Thick branching complex Severe/ papillae with intracellular and High-grade intraepithelial lumina, abundant eosinophilic (oncocytic) cytoplasm, large round nuclei with prominent nucleoli.

+

–

+

Reproduced from Furukawa et al., ref 12, with permission.

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a

b

c

d

Fig. 4.4 Photomicrographs showing morphological appearance of (A) gastric-type IPMN, (B) intestinal-type IPMN, (C) pancreatobiliary-type IPMN, and (D) oncocytic-type IPMN. (Images courtesy of Dr. T. Furukawa, Tohoku University School of Medicine, Sendai, Japan, from ref. 12, with permission.)

as the oncocytic subtype (12). This tumor has a similar gross appearance to conventional IPMN and is histologically characterized by stratified oncocytic epithelial cells with eosinophilic finely granular cytoplasm. Small intraepithelial mucincontaining “lumina” are often prominent (3, 13).

4 Differential Diagnosis Intraductal papillary mucinous neoplasm was often misdiagnosed as a mucinous cystic neoplasm (MCN) prior to the widespread recognition of IPMN in the late 1980s and early 1990s (14). IPMN often presents as a mucin-filled cystic mass like MCN and microscopically both lesions are lined by variably dysplastic, mucin secreting columnar epithelium. However, in MCN the papillae are generally less well developed and a characteristic cellular “ovarian” stroma is typically present (3, 9). Moreover, MCN usually occurs in women with a younger median age, are generally located in the tail or body of the pancreas, and rarely communicate with the pancreatic ductal system (4, 15). Differentiating between IPMNs and some pancreatic intraepithelial neoplasia (PanIN) can also be problematic given that both lesions consist of intraductal

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neoplastic proliferations of mucin-producing columnar cells with a variable degree of papilla formation (1). Although PanIN typically arise in the small ducts, they may occasionally involve larger ducts. Conversely, although IPMNs generally arise in the larger ducts, they can involve smaller ducts. Indeed, both lesions may coexist in the one pancreas, possibly accounting for the development of pancreatic ductal carcinoma several years after “curative” resection for noninvasive IPMN and the presence of ductal adenocarcinoma distant from an IPMN (5, 7, 16, 17). A consensus conference in 2003 proposed revised definitions for these lesions with PanIN being characterized as a microscopic lesion usually involving ducts less than 5 mm in diameter, while IPMN is typically a grossly visible lesion greater than 10 mm in diameter arising from the main pancreatic duct or branch ducts (1). These new guidelines have resulted in more consistent classification and nomenclature of these lesions, although some distinctions, particularly with regard to grading, remain problematic (18).

5 Cytologic Diagnosis The cytologic diagnosis of intraductal mucinous neoplasm has received increasing attention in the literature, particularly with more widespread use of endoscopicguided fine-needle aspiration cytology (EUS-guided FNAC) in the preoperative investigation of cystic pancreatic lesions (19–26). However, there is considerable overlap in cytologic features between IPMN and other pancreatic lesions, especially MCN and mucinous ductal adenocarcinoma (19, 20, 24), and hence FNAC should always be correlated with the clinical presentation and imaging appearances. Reported values for the sensitivity of EUS-guided FNAC for the diagnosis of IPMN vary from 38 to 100%. Likewise, there is considerable variation in reported specificity, in one series it was 98%, although in another there were five false-positive diagnoses out of 18 cases (19, 20, 22, 26). These variations probably reflect a number of factors including the skill of the endoscopic aspirator and adequacy of the sample, the experience of the cytopathologist, whether the cytopathologist attends the FNA procedure, the use of adjunct techniques such as liquid-based collection methods, and the rigor with which the clinicopathologic correlation is performed (20, 22, 24). The most consistent cytologic finding in IPMN is abundant thick colloid-like mucin, whereas the presence of papilliform fragments and intracellular mucin appear to be the most specific features (19, 20). Studies of the preoperative grading of IPMN utilizing cytology have demonstrated features that correlate with moderate dysplasia (tight epithelial clusters) or carcinoma in situ (abundant background inflammation and parachromatin clearing). The presence of necrosis has been found to be strongly suggestive of invasion (25). The sensitivity of EUS-guided FNAC for the prediction of malignancy in IPMN is ~60%, whereas the specificity is 100% (19, 21). However, the sensitivity of FNAC is significantly limited by the focal nature of malignancy in many IPMN with the attendant risk of sampling error leading to underestimation of tumour grade (19, 23).

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Brushing and biopsy specimens obtained during endoscopic retrograde pancreatography have also been utilized for the diagnosis and grading of IPMN with similar results to EUS-guided FNAC (23). Ultimately, the accuracy of cytologic diagnosis and its value as a tool in the preoperative assessment of IPMN is dependent on close correlation with the clinical and imaging information.

6 Frozen Section Diagnosis Frozen section examination has been advocated principally for assessment of the pancreatic transection margin during resection of intraductal papillary mucinous neoplasms. Recent studies have reported that routine frozen section examination during pancreatico-duodenectomy or distal pancreatectomy resulted in the detection of IPMN at the resection margin, followed by additional resection, in 23–30% of patients (27, 28). Discrepancy between frozen section and subsequent definitive paraffin section examination was low and there was no recurrence of noninvasive IPMN (27–29). However, given that a small proportion of IPMN are multifocal within the pancreatic duct system, inevitably some patients will still have undetected tumor in the pancreatic remnant even when the surgical resection margin is clear (4–6, 27).

7 Molecular Pathology of IPMN A better understanding of the molecular pathology of IPMN will assist in defining the natural history of these lesions, most importantly the risk of invasive malignancy, and facilitate the development of novel diagnostic and therapeutic strategies. IPMN demonstrate molecular genetic aberrations of some oncogenes and tumor suppressor genes common to premalignant lesions and cancers of other organs, including activating K-ras mutations, p53 mutation, cyclin D1 overexpression and loss of p16INK4A expression (30). Molecular aberrations common to IPMN, PanIN and invasive ductal adenocarcinoma include activating K-ras mutation (31), p53 protein accumulation (8, 31, 32), cyclin D1 overexpression, loss of p16INK4A expression (32), HER-2/neu overexpression and aberrant expression of oncofetal antigens such as CEA, CA19-9, and DUPAN-2 (33) have been identified in IPMN and invasive carcinoma. Chromosomal aberrations associated with IPMN and invasive carcinoma of the pancreas also share a similar profile (34, 35). Heterogeneity of allelic loss within IPMN lesions suggests progression within IPMN (34, 35), and as a consequence present further difficulties in developing diagnostic strategies. IPMNs do not commonly demonstrate microsatellite instability (36). Comparison of proliferative indices in IPMN with normal ductal cells and invasive cancer, using immunohistochemistry for Ki-67 and proliferating cell nuclear antigen (PCNA), demonstrates a progressive increase from IPMN adenoma to IPMN borderline, to

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IPMC, through to invasive carcinoma (8). Similarly, aberrations in cell cycle regulatory genes increase in frequency with increasing dysplasia in IPMN and appear to be similar to those in PanIN lesions with an equivalent degree of dysplasia (32). High telomerase activity is detectable in IPMC (37) and PC, as is COX-2 expression (38). Furthermore, PanIN lesions found in association with IPMN also demonstrate similar molecular aberrations (17). There are two notable differences in ductal adenocarcinomas that arise from IPMN compared to those that do not. First, approximately 40% of carcinomas arising from IPMN are of the mucinous noncystic (colloid) type with 60% being of the tubular type in contrast to carcinomas arising without IPMN where over 95% are of the tubular type (7, 9, 10). Second, 30 kg/m2 compared to individuals with a BMI 20 years (OR = 5.3; 95% CI 2.1–13.4) compared to individuals who did not smoke or smoked for 80%) of the analyzed cell lines fell into either G2 (50%) or G3 (33.3%) with only 16.7% of the pancreatic cancer cell lines identified as G1. Interestingly, this categorization does not parallel the same distribution seen in pancreatic adenocarcinoma tumors, which is 33% G1, 51% G2, and 16% G3 (17). This difference can be accounted for in the inherent difficulty in culturing cell lines from well differentiated tumors. Due to the possible limitations in the number of cell lines evaluated, the authors were not able

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to correlate the grade of the cell lines to K-ras, p53, p16, or DPC4/smad4 mutation rates. There was, however, a relationship between grade and population doubling time, with G1 cells having a significantly higher doubling time than G3 cell lines. Sipos et al also studied immunocytochemical features of monolayer cultures and spheroids generated from cultured pancreatic ductal adenocarcinoma cells stained for cytokeratin 7, 8, 18, and 19, vimentin, MUC1, 5, 6, carcinoembryonic antigen (CEA) and pancytokeratin. In vivo, the expression of cytokeratins 7, 8, 18, and 19 is a well-defined characteristic of pancreatic adenocarcinoma (18–21). In contrast, vimentin is a marker of undifferentiated carcinomas and is more frequently absent in differentiated PDAC in vivo (22). Based on their findings, vimentin was present in 10 of the 12 pancreatic adenocarcinoma cell lines studied as well as variable expression of cytokeratins 7, 8, 18, and 19. In addition none of the other markers evaluated (including MUC1, 2, 5, 6, CEA, synaptophysin, and chromogranin A) had a direct relationship with the grade of the cell lines (2). Therefore, these immunohistochemical markers do not seem to be useful for the categorization of pancreatic cancer cell lines with regard to their phenotypic qualities and differentiation status. The degree of differentiation or grade has been deemed as a surrogate for the aggressiveness of a tumor. A progression model for PDAC has been proposed with the classification of preinvasive ductal lesions called pancreatic intraepithelial neoplasia (PanIN) that are hyperplastic and progress through a series of architectural and cytological changes to become malignant (23). PanINs are subclassified into PanIN-1A, PanIN-1B, PanIN-2, and PanIN-3. However, the global definition of a PanIN is a microscopic papillary or flat, noninvasive, epithelial neoplasm arising in the pancreatic ducts. PanINs are characterized by columnar-to-cubical cells with varying amounts of mucin and degrees of cytologic and architectural atypia (24). PanINs usually involve ducts 75%), CDKN2A/INK4A (90%), and SMAD4/DPC4 (~55%) (17). MCNs share a largely overlapping spectrum and frequency of mutations with one notable nuance: mutations in SMAD4/ DPC4 are essentially invariant in invasive disease (18). Although IPMNs also possess frequent mutations in these cardinal genes, the reported ranges and specifics for these events are not as congruous as those for the other two routes (11). Moreover, IPMNs harbor several unique genetic events including mutations in PI3KCA (19) and LKB1/STK1 (~25%) (20), the gene associated with Peutz-Jeghers syndrome; similar mutations are not seen in PanIN-PDA or MCN-IDA. Finally, IPMNs very rarely mutate SMAD4/DPC4 (21) perhaps the clearest difference between this and the other two routes to invasive ductal cancers. The cystic neoplasms do share one important and distinguishing feature, however: they are often curable by resection. This finding is particularly intriguing for invasive MCNs as these neoplasms share virtually identical spectra and incidences of key mutations as PanIN-PDA. Moreover, the invasive disease that arises out of MCNs strongly resembles conventional ductal adenocarcinoma and is distinguished largely by the presence of associated cystic neoplasms. Thus, static analyses of resected tumor specimens do not resolve this paradox, although an important clue is provided by the essentially uniform occurrence of DPC4 mutations in invasive MCNs (discussed further in the following).

3 Brief History of Modeling in the Mouse Pancreas The long history of genetic manipulation in the mouse together with the widespread availability of inbred strains and, more recently, embryonic stem cell lines, make the laboratory mouse a particularly attractive vehicle for the study of disease pathogenesis. Interestingly, the murine pancreas, unlike that of the hamster and rat, has been relatively resistant to chemically induced carcinogenesis, a barrier that may have recently been surmounted (22, 23). Until recently, it has also resisted attempts to model ductal adenocarcinoma genetically. Among the pioneering experiments to introduce transgenes into the mouse genome were those linking the expression of presumptive oncogenes to pancreas-specific promoters (24–27). The transforming effects of c-myc (28), SV40 T and t antigens (29, 30), and HrasG12V (31) were probed in this manner and early models of acinar and islet cell neoplasia

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were developed, some of which continue to be studied today (32). More recently, Elastase promoter elements have been used to drive expression of TGFα (33–35) and K-rasG12D (36), whereas the ductal-selective CK-19 promoter has been linked to K-rasG12V (37), in model systems that achieved some notable similarities to the human disease. Each of these investigations has yielded critical insights into the functions of specific genes and the apparent requirements for specific cellular contexts and levels of gene expression to recapitulate histopathology. Collectively, they have also laid the foundation for subsequent efforts. The abilities to target the endogenous locus by homologous recombination in ES cells and to spatially, and even temporally, restrict gene expression have led to a new generation of mouse models of human disease (38, 39). Efforts to model pancreas cancer have also benefited from these technologies. Although the considerable differences in biology and physiology (40) should necessarily give pause to scientist and physician alike, carefully conceived and constructed animal systems can help illuminate and clarify otherwise obscure questions on mechanisms of human disease. Indeed, informed syntheses of clinical, biological, histologic, and molecular data from cognate human and animal diseases may hold the greatest promise to unravel heretofore intractable questions of human disease. The studies of human PDA described above have raised a number of pivotal questions, questions that have been or promise to be illuminated by investigations in animal systems: 1. Are PanINs bona fide precursors to invasive PDA? 2. Is there a temporal relationship between the various putative stages of PanIN lesions? 3. Do activating mutations in K-ras initiate disease? 4. Which compartment(s) is competent to initiate disease? In other words, what is the cell-of-origin of PDA? 5. Which, if any, of the identified mutations in the principal tumor suppressor gene pathways play a causal role in disease progression? 6. Do different combinations of mutations define distinct histopathologic forms of the disease? 7. Does the sequence in which the mutations arise help define the ensuing disease? 8. At what point along the continuum of preinvasive disease does invasion pass from the possible to the inevitable? In other words, at what point must we intervene?

4 Pancreas Cancer: Ras Reveals the Way The earliest genetically engineered mouse models involved targeted deletion or “knock-out” of prominent tumor suppressor genes, including those frequently mutated in PDA such as Trp53 (41, 42), Cdkn2a/Ink4a (43), and Smad4/Dpc4 (44–47). None of these animals revealed any evidence of pancreatic pathology, however.

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Fig. 10.2 Histologic progression in genetically engineered murine models of pancreatic ductal adenocarcinomas. Early preinvasive, advanced preinvasive (carcinoma in situ), invasive and metastatic neoplasms are shown from K-ras G12D/+;Cre (A–D) (47, 49), K-rasG12D/+;Trp53R172H/+;Cre (E-H) (49) and K-rasG12D/+;Dpc4flox/+;Cre (I–L) (53) animals. A PanIN-1A lesion (A) denotes a transition from the simple cuboidal epithelium of a normal duct to a columnar phenotype with abundant apical mucin. In a PanIN-3, or carcinoma-in-situ (B), complete loss of polarity, nuclear atypia, and clusters of luminal cells are observed. Progression to invasive disease from these precursors can reveal moderately well-differentiated (C) to poorly differentiated to sarcomatoid or spindle cell morphologies. Metastatic disease (D, asterisk), in this case to the diaphragm (d), typically retains the morpholigcal features of the primary malignancy. The preinvasive (E, F), invasive (F), and metastatic (H, asterisk) disease in K-rasG12D/+;Trp53R172H/+;Cre animals also follows the classical PanIN-to-PDA progression with a preponderance of well-differentiated histology. The cystic lesions arising in K-rasG12D/+;Dpc4flox/+;Cre mice reveal all of the diagnostic features of human MCNs including a columnar, mucin-filled epithelial lining without significant papillary projections into the lumen (I, arrows). These lesions progress through increasing degrees of dysplasia from low-grade (I), to moderate grade, to high-grade (J; microscopic foci of invasion are also evident, arrows). Invasive disease (K, asterisk) reveals a glandular histology in clear association with a mucinous cystic neoplasm (arrow). Once again, the metastases arising from invasive MCNs (L, arrows), here shown in the liver, retain the cystic morphology of their primary cancers, including recapitulating varying degrees of cellular and nuclear atypia

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These studies suggested that an oncogenic mutation may serve as the rate-limiting event and therefore be required to initiate disease. Because embryonic expression of the activated K-ras allele is a lethal event (48), studying the potential tumorigenic effects of oncogenic K-ras requires targeted expression to specific tissues postgastrulation. This can be achieved through conditional expression systems using CreLox (49) or Flp-Frt (50) technologies. Using these strategies, the physiologic expression of oncogenic K-rasG12D targeted to the Pdx-1 and p48-expressing compartments of the developing murine pancreas resulted in the faithful recapitulation of all three postulated stages of preinvasive lesions, or PanINs (51). These lesions progressed over time, accumulating increasing numbers and degrees of cellular and nuclear atypia, ultimately culminating in the development of invasive and metastatic PDA (Fig. 10.2). In addition, a number of signaling pathways were aberrantly activated at even the earliest stages of preinvasive disease, mirroring their behavior in the human disease and implicating oncogenic K-ras in their dysregulation. These pathways further suggest potential targets for chemoprevention and treatment. Finally, a distinctive signature in the low molecular weight serum proteome of animals was preinvasive disease was identified, providing proof-of-principle of the existence of definable markers for the earliest stages of disease.

5 Tumor Suppressor Gene Mutations Shape What K-ras Begins Similar targeting strategies have been used to systematically evaluate the effects of the other genetic events of postulated importance, both alone and in combination. Unlike tumor suppressor gene mutations commonly encountered in human malignancies, which generally involve genomic deletion and/or epigenetic silencing, mutations in TP53 found in PDA invariably involve point mutation of one allele at one of several “hot spots” followed by LOH of the locus (52). Engineered mutations of one of the most frequently encountered point mutations in human PDA, Trp53R172H (corresponding to TP53R175H), when concomitantly expressed with KrasG12D, results in the accelerated evolution of disease with highly glandular histology and widespread metastases (53) (Table 10.2). The histological phenotype as well as the sites and frequencies of metastases all closely recapitulate the human disease. Intriguingly, the primary cells isolated from pancreatic tumors and metastatic lesions exhibit high degrees of both simple (numerical) and complex (structural) instability, notable features of human PDA, perhaps reflecting a gain-of-function phenotype associated with point-mutation as opposed to deletion of Trp53. Disease progression also invariably involves LOH of the Trp53 locus, again as is seen in human PDA. Interestingly, rare variants including adenosquamous, sarcomatoid and cystic pathology were also seen again at similar frequencies encountered in human pancreatic cancer. These findings speak to the considerable plasticity exhibited by the target cell in response to a defined set of genetic perturbations and implicate, though by no means prove, a mutated pluripotent progenitor cell as the cell-of-origin. Thus, it appears that the combined physiologic expression of oncogenic

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Table 10.2 Phenotypes of genetically engineered mouse models of pancreatic ductal carcinoma Primary disease Genotype

Preinvasive

Invasive

Histology

Median survival (mo)

References

K-rasG12D/+ K-rasG12D/+;p53R172H/+ K-rasG12D/+;p16/p 19fl/fl K-rasG12D/+;p53flox/+; p16(+/–) K-rasG12D/+;Dpc4fl/+ K-rasG12D/+;Dpc4fl/fl K-rasG12D/+;Dpc4fl/fl K-rasG12D/+; p16/p19fl/fl;Dpc4fl/fl K-rasG12D/+;Tgfbr2flox/flox K-rasG12D/+;CLEG2

PanIN PanIN PanIN

PDA PDA PDA

G, S G G, S, A

15 5 2

57, 53 53 54

PanIN

PDA

G, S

3.5

56

MCN MCN IPMN PanIN> IPMN PanIN PanIN + “lobular”

IDA IDA PDA PDA

C + G + OS C + G + OS C+G G>S

15 8 4 3.5

57 57 58 58

PDA Undiff. carcinoma

G>S U

2 1

62 75

A, anaplastic; C, cystic; G, glandular; OS, ovarian stroma; S, sarcomatoid; U, undifferentiated.

Fig. 10.3 Genotype determines phenotype in mouse models of invasive pancreatic ductal adenocarcinoma. K-rasG12D/+;Cre (47, 49) and K-rasG12D/+;Trp53R172H/+;Cre (dorsal view) (49) mice typically develop large, firm tumors at the head-of-the-pancreas (asterisks) frequently accompanied by metastatic disease (arrows), here shown to the liver. In contrast, K-rasG12D/+;Dpc4flox/+;Cre animals developed multilocular cysts (arrows) in the body and tail of the organ, usually filled with serous fluid. The head of the pancreas was frequently micronodular (arrowheads), but lacked a dominant mass or the large cysts noted in more distal portions of the gland

K-rasG12D and Trp53R172H faithfully recapitulates the human disease at the molecular, cellular, and organismal levels, including the rare occurrences of alternative histologies. Moreover, the absence of cooperating mutations in the other principal tumor suppressor pathways implicated in pancreatic cancer pathogenesis, led to the postulation that distinct genetic pathways to PDA may exist with unique biological behaviors (Fig. 10.3). The possibility of discrete genetic subtypes of PDA has

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potentially important implications for disease detection, treatment and chemoprevention strategies (discussed further in the following). Biallelic deletion of the contiguous Cdkn2a/Ink4a (p16) and Cdkn2d/Ink4d (p19) loci also hastens disease progression, resulting in a very aggressive local disease with rare microscopic metastases. Interestingly, these tumors frequently displayed sarcomatoid histology, often as the predominant pattern (54, 55). Subsequent studies confirmed this tendency of p16 mutations, in particular, to steer pathology strongly toward sarcomatoid, or spindle cell, carcinomas (56). The uniform sarcomatoid histology seen with p16-null animals is pulled toward glandular, and secondarily anaplastic phenotypes with heterozygous mutation of Trp53; homozygous mutation of both p16 and Trp53 appears to further shift some of the glandular tumors toward an anaplastic phenotype, thought to represent a particularly aggressive form of the disease. Compound heterozygous mutation of p16 and p19 similarly results in a modest shift toward glandular phenotypes. Conversely, the strong tendency toward glandular histology seen with mutation of Trp53 is shifted toward an anaplastic pattern in animals with compound homozygous mutation of Trp53 and p16. In each of the mentioned models, mutation of additional tumor suppressor gene pathways was not observed, suggesting that mutation of the Ink4a and Trp53 pathways represent distinct genetic routes to PDA with definable clinical and histological characteristics. The notion of different genetic routes to PDA was further supported by the identification of distinct clusters of copy number alterations segregating tumors that developed in the context of different TSG mutation combinations (56). From here, studies logically turned to the next most frequently altered signaling pathway in human PDA, the TGFβ cascade. The findings with mutation of Smad4/ Dpc4 in the setting of K-rasG12D were unexpected and startling, although in some respects, clues from human disease progression were perhaps telling.

6 TGFβ Signaling and Cystic Neoplasia: When Matters as Much as Which As noted, mutations in SMAD4/DPC4 occur in about half of PanIN-PDA and almost uniformly in MCN-IDA, consistent with a relatively late occurrence in the progression of PanINs while suggesting a potentially earlier event in the progression of MCNs (i.e., mutation of the first allele occurring relatively early allowing sufficient time for mutation of the second allele in virtually all cases). Smad4 is an essential gene in the mouse, although pancreas-specific deletion does not cause any overt developmental or functional abnormalities (57, 58). Consistent with the role of K-ras mutation as the rate-limiting event in pancreatic tumorigenesis, pancreatic pathology only becomes manifest in the setting of concomitant expression of KrasG12D resulting in the development of cystic neoplasms. Although Bardeesy et al. described the presence of IPMNs (58), Hingorani and colleagues found that MCNs developed (57). The precise reasons for this difference in observed pathology is unclear, but may have to do with differences in background strains (FVB in the

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Preinvasive Disease Advanced

Early

p53R172H

LOH

PDA

CIN p16/p19 Terminal Ductules

Tgfbr2 K-ras

PanIN Dpc4+/-

PDA PDA (mice); Medullary CA (humans) p16

-/1 Dpc4

MCN

IDA p53

Main/Branch Ducts

K-ras? PIK3CA? 1

IPMN2

LKB1?

p16? p53?

Colloid CA; IDA

The cellular pathway by which MCNs arise remains controversial, but see (57)). 2The development of IPMNs has also been reported in mice expressing oncogenic K-ras and deletion of Dpc4 (54).

Fig. 10.4 Pathways to pancreatic ductal cancers. A combination of studies on human and genetically engineered malignancies of the pancreatic ductal epithelium suggest distinct histologic and genetic trajectories to invasive disease with corresponding differences in clinical behavior and prognosis (see text for details)

former and mixed 129Sv/Bl6 in the latter) and/or differences in the specific targeting strategies used for Smad4. Heterozygous deletion of Smad4 in the context of endogenous K-rasG12D expression results in the elaboration of MCNs with distinctive ovarian stroma (see Figs. 10.3 and 10.4) (57). Thus, Smad4/Dpc4 manifests haploinsufficiency in constraining against the development of MCNs in the setting of oncogenic K-rasG12D. The proliferation of ductal cells in MCNs is also somewhat attenuated as compared with the more conventional PanIN counterparts. The murine MCNs progress to invasive mucinous cystadenocarcinomas (or invasive carcinomas in association with MCNs, in the more recent nomenclature). Progression invariably involves LOH of Smad4/ Dpc4, as is seen in the human disease, along with mutation of either Trp53 or p16Ink4a, but not both. Expression of p16 is extinguished through a combination of genomic deletion and promoter methylation, again as is most common in human pancreatic ductal cancers, whereas specific expression of p19 was retained in all but one case. Thus, in the end, the invasive carcinomas that arise through this histopathologic route acquire the same overall genetic repertoire as conventional PDAs. Intriguingly, K-rasLSL-G12D/+;Dpc4flox/+;p48Cre/+ (KDC) animals manifest a lower burden of metastatic disease and live longer than K-rasLSL-G12D/+;Trp53LSL-R172H/+;Cre (KPC) animals, which develop conventional PanIN-PDA. The same patterns hold true for their respective human correlates.

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The molecular and cellular properties of primary cells isolated from the tumors and metastases that develop from each route to ductal cancer help explain the profoundly different clinical and biological behaviors (57). First, KDC primary ductal cells have lesser degrees of both numerical and structural genomic instability than KPC cells. Second, their responses to TGFβ are tellingly distinct. The relationship of TGFβ to tumorigenesis is multifaceted and dynamic, affecting both cell autonomous and non-cell autonomous processes and shifting between roles as deterrent and guardian to instigator and accomplice (59–61). The role of TGFβ similarly evolves during the course of normal development, promoting proliferation and migration in the forming embryo, while exerting growth constraints and regulating homeostasis in adult tissues. The migratory and invasive impulses stoked by TGFβ in development are re-awakened during the course of oncogenesis and TGFβ can encourage epithelial-to-mesenchymal transition (EMT) and motility in cancer cells. Indeed, KPC ductal cells underwent dramatic EMT and became much more motile in response to TGFβ, consistent with their relatively aggressive clinical behavior. KDC cells on the other hand, were clearly resistant to these effects and, in some cases their migration was even inhibited by TGFβ. The tumor-promoting effects of TGFβ appeared to require intact Smad4 signaling. These data suggest a potential explanation, therefore, for the profoundly different biological behaviors of adenocarcinomas that arise from PanINs as opposed MCNs. K-rasG12D initiates preinvasive ductal disease. Early mutation of Smad4 shapes the subsequent differentiation of these lesions toward MCNs and slows their proliferation, while also predisposing them toward developing early LOH of the locus. In the absence of Smad4-dependent signaling, these cells resist the tumor-promoting effects of TGFβ, which not coincidentally, is often elevated in pancreatic cancers. Disease progression is eventually accompanied by additional mutations in either Trp53 or p16, resulting in carcinomas with a diminished capacity to invade and metastasize and, therefore, an increased likelihood of being cured by surgical resection. If on the other hand, mutations in either Trp53 or p16 occur next after K-ras-initiated preinvasive disease, the lesions are hastened down the conventional PanIN-PDA route. Along the way, the cells remain susceptible to the tumor-promoting effects of TGFβ, allowing them to undergo EMT and enhanced migration. Mutation of Smad4 at this relatively late juncture appears unable to significantly alter the differentiation state of these cells and may instead serve primarily to relieve a remaining restraint against uncontrolled proliferation. Thus, the inherent duality of TGFβ signaling is played out in these two phenotypic routes to PDA, which otherwise share the same overall complement of critical genetic mutations. While the working model suggested by these findings should serve to frame further inquiry and will undoubtedly be augmented by a more nuanced understanding over time, it also provides some readily testable hypotheses, a few of which have already been addressed in contemporaneous studies. For example, the demonstrated haploinsufficiency of Smad4/Dpc4 in inhibiting MCN formation presumably results from an imbalance between receptor Smad-dependent and -independent, or specific Smad4-dependent and -independent mechanisms of TGFβ signaling.

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In either event, mutation of the pathway further upstream, at the level of surface receptors for TGFβ, for example, would be expected to affect each of the arms equally and therefore not result in the altered differentiation state described above. In addition, concomitant or prior mutation of Trp53 or p16Ink4a followed by mutation of Smad4/Dpc4 would be expected to shift the phenotype toward conventional PDA. Although TGFBR1 and TGFBR2 are only very rarely mutated in human PDA (52), expressing the aforementioned K-rasG12D allele in the context of heterozygous Tgfbr2 mutation in the mouse did result in the development of conventional PDA and not cystic neoplasia (62), as predicted. Expression of K-rasG12D together with targeted biallelic deletion of Tgfbr2 hastened the development of well-differentiated PDA with rare metastases. Once again, mutations in the cardinal TSGs were not observed, although p16Ink4a levels varied and p19Arf protein expression was absent, despite the presence of specific message in most cell lines. Trp53 could be induced by ionizing radiation, consistent with an intact pathway. The longer tumor latency observed in Tgfbr2flox/+ mice was associated with a correspondingly increased metastatic burden. Interestingly, the primary and metastatic cells from Tgfbr2flox/+ animals retained the remaining wild-type allele, suggesting haploinsufficiency of Tgfbr2 in preventing pancreatic tumor progression in the context of oncogenic K-rasG12D. Moreover, some of the tumors in Tgfbr2 heterozygous mutant animals displayed sarcomatoid features, which were not seen in Tgfbr2flox/flox mice and which the authors therefore suggest may reflect a requirement for some preserved Tgfbr2 signaling to permit EMT to occur. The molecular and cellular responses to TGFβ of the tumor cells from these latter models were not described. A separate model employing the same conditional K-rasG12D allele in the context of a distinctly targeted Smad4 gene was also recently reported (58). Cystic neoplasms were again described, although they were characterized as IPMNs and not MCNs. All K-rasLSL-G12D/+;Dpc4flox/flox;p48Cre animals (n = 12) in this study developed IPMNs, although only two (17%) developed invasive disease. Additional heterozygous mutation of the combined p16Ink4a/p19Arf locus resulted in an increased proportion of animals with invasive ductal adenocarcinoma (12/13), although only a minority of these occurred in the setting of an IPMN (4/13), and no colloid carcinomas were described. In each of three cell lines from invasive tumors, LOH of p16Ink4a/p19Arf was observed. Finally, none of four animals with p48-targeted biallelic deletion of p16Ink4a/p19Arf loci and homozygous mutation of Dpc4 developed IPMNs, whereas all succumbed to invasive PDA. Thus, a shift toward conventional PDA was seen in the setting of concomitant mutation of the p16Ink4a/ p19Arf locus and Smad4, as predicted by Izeradjene et al. (57). Indeed, concomitant homozygous Smad4 mutation also appeared to shift the frequently sarcomatoid features observed with p16Ink4a/p19Arf mutation toward the more glandular histology observed in human PDA. In preliminary analyses of K-rasLSL-G12D/+;Trp53LSLR172H/+ ;Dpc4flox/+;p48Cre/+ animals, we too have observed a shift toward conventional PDA (K. Izeradjene, P. Calses, S. Hingorani, unpublished observations). Bardeesy and colleagues also observed a requirement for Smad4 signaling to mediate TGFβ-induced migration and EMT (58). They described a further association between histologic differentiation and response to TGFβ in the setting of intact

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Smad4. Specifically, tumor cells derived from undifferentiated carcinomas displayed increased migration and proliferation, whereas those from more welldifferentiated tumors were growth inhibited, less motile, and underwent apoptosis in response to TGFβ. Thus, several important unifying themes have emerged from these three studies on TGFβ-mediated signaling in pancreatic tumorigenesis, whereas reconciling the remaining notable differences promises to further illuminate the complex role of this important pathway.

7 Context and Chronology The collective findings from the more recently described genetically engineered mouse models of pancreatic cancer reveal unique and exquisite sensitivities of different cellular compartments and discrete regions of the pancreas to specific genetic perturbations, and affirm the role the local microenvironment plays in shaping the course of disease evolution. The targeting strategies used in these animal models introduce the respective mutations in all cells throughout the length of the organ. Nevertheless, K-rasLSL-G12D/+; Cre (51) and K-rasLSL-G12D/ + ;Trp53R172H/+;Cre (53) animals, for example, develop aggressive invasive ductal adenocarcinomas occurring primarily at the head of the pancreas. Concomitant expression of K-rasG12D and Smad4/Dpc4 mutation, on the other hand, instead induces cystic neoplasms (57, 58), most dramatically in the tail and body of the pancreas (57), which progress more slowly to invasive carcinoma. Moreover, in each of these models, the acinar and islet cell compartments appear to be largely unaffected, suggesting potential mechanisms of resistance to the transforming effects of oncogenic Ras based on differentiation program. The dramatically different biologies in the setting of intact versus haploinsufficient Dpc4 may reflect unique factors in the periampullary microenvironment, as compared with more distal regions of the pancreas, and concomitant differences in the abilities of these environments to cooperate with specific genetic events. The distinct phenotypes may also be the result of intrinsic differences in the tissue progenitor cells along the length of the ductal tree. The major portions of the pancreas evolve from different embryologic structures, experiencing a distinct repertoire of developmental influences that may imprint themselves on cells of the adult organ. The dorsal and ventral buds, which later fuse to form the mature organ, contribute disproportionately to the three major regions of the gland (63–65). The head of the pancreas is comprised of the ventral pancreas and a portion of the dorsal pancreas nearest the duodenum; the remainder of the dorsal pancreas alone constitutes the body and tail of the mature organ. A number of non-cell autonomous or cell-autonomous factors, therefore, may contribute to the unique sensitivities of distinct regions of the pancreas to specific genetic events and result in the elaboration of disparate pathologies. These possibilities are, of course, not mutually exclusive. The isolation and characterization of the cell-of-origin for pancreatic cancer is likely to profoundly influence strategies to detect and treat the disease; the search may

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ultimately involve the identification of different populations for each pathogenetic route to PDA.

8

Oncogeny Recapitulates Ontogeny

With rare exception, oncogenic mutations are not found in heritable cancer syndromes. None of the major activating mutations in K-ras2 found in PDA have been identified in the germline of patients (although recently less potent mutations in this proto-oncogene have been associated with Noonan syndrome) (66, 67). In the mouse, both germline deletion of K-ras and constitutive expression of K-rasG12D cause embryonic lethality, suggesting profound perturbations of developmental pathways. Perhaps not surprisingly, a number of signaling pathways critical for the development of the pancreas and related foregut organs are aberrantly regulated during the course of pancreatic cancer progression. For example, Notch signaling, which is essential for normal development of the pancreas, becomes quiescent in the adult organ (68, 69). The Notch pathway is aberrantly activated, however, in both human (70) and murine PDA (51, 57). Hedgehog (Hh) signaling, on the other hand, must be suppressed during development in order for the pancreas to be elaborated from foregut anlage, which otherwise develop into liver and intestine (71). Increased expression of soluble Hh ligands and evidence of pathway activation also occurs in both human (72, 73) and murine pancreatic cancer (51, 53, 57), and ectopic pancreatic expression of Sonic hedgehog results in the formation of tubular complexes resembling human PanINs (73, 74). Targeting these developmental pathways may also be of therapeutic value, as Hh inhibition slows the growth of xenografted human pancreatic cancer cell lines (72, 73). Recent studies of cell autonomous activation of Hh signaling have extended these findings (75). Pancreatic expression of a constitutively active Gli2 transcription factor resulted in the development of undifferentiated carcinomas in about one third of the animals, albeit not through conventional PanINs as no such preinvasive lesions were observed. When expressed in the context of the conditional K-rasG12D allele described in the preceding, normal pancreatic architecture was largely disrupted and replaced by fibrosis and the appearance of “lobular structures.” These animals did develop preinvasive ductal lesions and did so more rapidly than KCre animals; approximately 60% of the animals developed undifferentiated carcinomas, although no conventional ductal adenocarcinomas were observed. Thus, constitutive Hh signaling in the epithelial compartment accelerated the development of preinvasive lesions and increased the likelihood of developing undifferentiated carcinomas. However, given that animals expressing activated Gli2 alone also developed undifferentiated tumors in the absence of PanINs, it remains formally possible that the carcinomas seen in the latter cases also arose through an alternative mechanism. Nevertheless, these experiments further underscore the relationship between Hh signaling and oncogenic K-ras in pancreatic tumorigenesis. It would be of interest to see if targeted deletion of Hh pathway constituents, or Notch components, in the context of endogenous K-rasG12D, influence tumor progression.

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9 The Biology of RAS: Sometimes Less Is More Genetically engineered mouse models involving targeted expression of K-ras have also added critical and nuanced insights into the basic biology of Ras signaling. The past 30 years of experimentation on Ras biology have amply described the phenomenon of “premature senescence” induced by oncogenic Ras in the absence of cooperating oncogenes or mutations in critical tumor suppressor genes, such as TP53 or CDKN2A/INK4A (76–79). Not denying the profound importance of these seminal studies to current understanding in cancer biology, these findings have also tended to belie the considerable genetic epidemiologic evidence suggesting that mutant Ras acts as an initiating event in tumorigenesis. Indeed, in pancreatic cancer, Ras mutation is the earliest and most frequent major genetic event encountered (9, 80). How does one reconcile these two disparate conclusions? More recent studies have revealed the exquisite sensitivities to both the levels and cellular context of oncogenic Ras signaling in determining the precise physiological response. Thus, endogenous levels of activated Ras appear to induce widespread cellular proliferation in many (48), although not all (81), cellular contexts. The senescence response, in turn, is activated in some contexts and not others, and by differing contributions from several key signaling pathways, including for example Trp53/p19Arf, p16Ink4a, and MAPK. Thus, in the absence of additional mutations in critical tumor suppressor genes, transfected Ras expressed from a heterologous promoter indeed induces senescence, through activation of safeguarding tumor suppressor pathways. However, when expressed from its own promoter at physiological levels, as presumably occurs with spontaneous mutations of the locus, activated Ras instead induces proliferation in a number of cellular compartments. Thus, although there appear to be very real differences in the precise genetic requirements for transformation in human and murine cells (82– 84), the requisite levels of oncogenic ras are not likely to be among them.

10 Found in Translation: Moving from Cage to Clinic The current standard of care for advanced PDA is infusional gemcitabine, a deoxycytidine analog that provides only a very modest survival benefit and some symptomatic improvement in a minority of patients (85). Attempts to augment this regimen with platinum or fluorouracil, agents effective in other gastrointestinal malignancies, and to modify the dose, rate and route of administration of specific agents have met with limited success (86–88). Combined treatment with Erlotinib, an EGFR antagonist, represents an incremental advance over gemcitabine alone without significantly changing the overall outcome of the disease (89). The study of novel investigational agents and approaches for pancreatic cancer is hampered by the difficulties inherent in identifying sufficient numbers of patients with advanced diseased that are nevertheless healthy enough to meet enrollment criteria.

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a t1

t2

t3

t4 K-ras G12D

b t1 K-rasG12D;p16/19fl/fl

t1

t2 K-rasG12D;p53R172H/+

t1

t2

t3 K-rasG12D;Dpc4fl/fl

Fig. 10.5 The use of genetically engineered mouse models of pancreatic ductal adenocarcinoma in preclinical studies. Each of the described genetic models of disease has distinct tumor latencies, spectra of locally invasive and metastatic disease burden, and life expectancies. Therefore, the models lend themselves to a wide range of potential preclinical applications, including investigations of markers for early detection, agents for chemoprevention and treatment, and markers of disease response. Biospecimen collections, imaging studies, and drug administrations can be performed at defined intervals depending on the aims of the experiment and the clinical characteristics of the chosen disease model

In the end, the most relevant measure of animal models of human disease, and their greatest potential value, lies in their ability to impact the nature and delivery of clinical care. Specifically, will the mouse models described here prove to be more predictive of the human disease than previous preclinical models? The proof is in the testing. The development of distinct murine models of genetic subtypes of PDA (Fig. 10.5) with apparently unique phenotypes and latencies enables a wide range of investigations into disease detection, treatments, and markers of response. Moreover, if indeed these unique genetic and histopathologic routes to invasive ductal adenocarcinoma of the pancreas have direct equivalents in patients, then each type is also likely to require its own strategies for detection and therapy. As discussed, mice expressing targeted endogenous K-rasG12D develop the full spectrum of PanINs which slowly progress to invasive and metastatic disease (51) (see Fig. 10.5). Thus, this model is ideally suited for studies of early detection of preinvasive disease and of potential chemopreventive agents, for example, which would be particularly appropriate for high-risk cohorts of patients with familial pancreatic cancer

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(90, 91). Disease and target response would most likely be assessed by pathology at a defined study endpoint. Survival studies, on the other hand, could more readily be performed in models of rapidly advancing disease (53, 54, 62) (see Fig. 10.5). Moreover, the different propensities for locally advanced and widely metastatic disease in these models may distinguish the abilities of different agents to target local versus systemic disease burdens.

11 Conclusions and Future Directions The last several years have seen considerable progress in the faithful modeling of diverse histologic and genetic pathways to invasive ductal cancers of the pancreas. An emerging theme from these models reveals the essential role of oncogenic K-ras in initiating preinvasive disease and the existence of discrete combinations of additional mutations in tumor suppressor genes that influence the differentiation pattern and clinical behavior as disease progresses. Thus, each of the major tumor suppressor pathways critically shapes the course of neoplastic evolution in the pancreas in response to the challenge of an oncogenic K-ras stimulus. Similar approaches can, and undoubtedly will, be used to explore the effects on disease initiation and progression of genetic perturbations of Notch pathway members, Wnt/β-catenin pathway components, matrix metalloproteinases, and so on in the context of K-rasG12D expression. Recently developed murine models of heritable forms of pancreatitis (92, 93) may also be used in conjunction to study the influence of inflammatory injury in promoting K-ras-initiated disease, perhaps illuminating mechanisms behind the increased risk of pancreas cancer in patients with chronic pancreatitis (94, 95). Inflammation may induce genetic damage directly, stimulate proliferation of tissue progenitor cells, and/or increase the receptivity of specific cellular compartments to the transforming effects of mutant Ras. Finally, combining inducible systems should enable more precise control of the timing of various genetic perturbations, revealing the impact of temporal sequence on shaping the resultant disease. With an expanding array of tools now at hand, the field is poised to make decisive inroads into this formidable disease, transforming fear into hope and bracing humility with confidence. Acknowledgments Work in my laboratory is supported by CA114028, P30 CA15704, AACRPanCAN Career Development Award, Canary Foundation, Mead Foundation and Rosenzweig Foundation.

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Chapter 11

Molecular Signaling Pathways in Pancreatic Cancer Genevieve M. Boland and Sarah P. Thayer

1 Pancreatic Cancer: Background and Clinical Significance The pancreas is an extremely intriguing organ that has been the topic of discussion and study since its first description by the Greek anatomist and surgeon Herophilus around 300 BC. It is a retroperitoneal gland involved in various processes of human physiology, including blood glucose homeostasis, and secretion of digestive enzymes. In the context of the field of oncology, cancers of the pancreas can be divided into those of exocrine and those of endocrine lineages. The most common tumor of the pancreas is pancreatic adenocarcinoma, responsible for an estimated 37,680 newly diagnosed cases in 2008 with an associated 34,290 deaths from the disease in the same year (www.cancer.org). These numbers are a staggering example of the poor prognosis associated with this disease.

1.1

Definition of Pancreatic Adenocarcinoma

There are many signaling pathways implicated in the development and evolution of pancreatic adenocarcinoma. The role of these signature signaling pathways in driving specific biological behaviors during pancreatic cancer initiation, progression, and migration remains to be defined and thoroughly clarified. Pancreatic cancer is known for its characteristic histologic, genetic, and biologic phenotypes (1–7). Histologically, pancreatic adenocarcinomas are composed of neoplastic cells that demonstrate glandular or ductal differentiation, which has been known to take on many gastrointestinal characteristics (8–11). Pancreatic cancer is defined not only by its abnormal epithelium but also by its extensive non-neoplastic desmoplastic response. This extensive tumor stroma is composed of myofibroblasts, lymphocytes, extracellular matrix (ECM) proteins such as collagen, as well as trapped nonneoplastic pancreatic tissues such as islets. Pancreatic ductal adenocarcinomas (PDACs), unlike pancreatic intraepithelial neoplasias (PanINs), demonstrate an infiltrative growth pattern. The biology of pancreatic cancer is distinguished by its early dissemination even with small primary tumor loads. It also is known for its

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propensity for early perineural and peritoneal metastases (3, 12–14). Another wellknown biologic behavior is the ability of these cancers to be highly resistant to chemotherapy and radiation (15–17). Genetically this tumor is noted to be markedly aneuploid, with extensive ongoing chromosomal instability. The molecular mechanisms that control these behaviors are just beginning to be identified and understood, with the eventual goal of regulating these various cellular behaviors through signaling mediators and/or inhibitors.

1.2 History of Pancreatic Signaling Research Due to the devastating natural course of pancreatic adenocarcinoma, much energy has been focused on the identification, classification, and biology of this cancer (1–7). A series of precursor lesions have been identified that correlate with various stages of cancer development. Pancreatic cancer is believed to arise from histologically well-defined precursor lesions termed pancreatic intraepithelial neoplasias (PanINs). In this progression model, the normal cuboidal ductal epithelium is replaced by progressive histologic lesions: PanIN-1A, PanIN-1B, PanIN2, and PanIN3 (carcinoma in situ) (18). The key features noted in the sequential progression of PanIN lesions are enhanced mucin production, graded nuclear atypia, and loss of cellular polarity, with increasing abnormality of ductal structures. Additionally, the rate of cellular proliferation increases with advancing stages of PanINs as well as with increased aggressiveness of tumor behavior (18–22). These progressive histologic changes have been correlated with progressive genetic mutations and signaling alterations. During the earliest stages of PanIN formation (i.e., PanIN-1A and PanIN-1B), inciting oncogenic events are thought to be mediated by activating mutations of K-ras (23–25), as well as telomere attrition (7, 26) and loss of p21 (7, 21). Increased EGF signaling, such as in the context of overexpression of Her-2/neu, has also been correlated with these early neoplastic changes (27). By the PanIN-2 stage, an intermediate stage in lesion progression, there is additionally the loss of p16 (22, 28–30) and loss of PTEN (31, 32). In the setting of PanIN-3 (or carcinoma in situ), epithelial cells now demonstrate increased expression of receptor for various signaling pathways. There is an up-regulation of the EGF receptor as well as c-Met (the HGF receptor), concurrent with changes in TGF-β receptor expression level (27, 33–42). The loss of Smad4 (DPC4), an intracellular mediator of TGF-β signaling, also compounds the down-regulation of TGF-β receptors and enhances the TGF-β resistance of these oncogenic cell populations (39, 43–46). Cell cycle regulators such as p53 and BRCA2 are often mutated or inactivated in more advanced stages of tumorigenesis (Fig. 11.1) (47–50). All these molecular events have been catalogued and recognized as integral in establishing the evolution and progression of pancreatic adenocarcinomas. The identification of common mutation patterns suggests that these factors may play key roles in the formation of this tumor as well as may represent common mechanisms of lesion development (2, 51, 52).

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Fig. 11.1 Pancreatic adenocarcinoma: cell-cell and cell-matrix signaling. During the progression of normal ductal epithelium through the stages of PanIN progression and finally to the stage of invasive adenocarcinoma, there are many mutations and alterations in signaling cascades that affect not only the ductal epithelial component, but also the stromal component of the pancreas. This results in the robust desmoplastic reaction characteristic of pancreatic adenocarcinomas. These changes correlate with specific changes in signaling molecules and functional behavior of the epithelial cells themselves. These alterations in local cytoarchitecture contribute to the progressive development of tumors, as previously described. Listed here are known changes associated with these various premalignant and malignant stages in pancreatic cancer formation

1.3

Desmoplasia and the Dynamic Cross-Talk Between the Stroma and the Epithelium

It is well documented that many carcinomas induce a modified stroma during tumor progression. Pancreatic adenocarcinoma is particularly notable for the activation of the stromal compartment into a very robust desmoplastic reaction (8–10). These stromal changes have been ascribed to the induced expression of various growth factors that promote angiogenesis, altered ECM protein expression, increased fibroblast proliferation, and inflammatory cell recruitment. Subsequent studies of this process of tumor–stromal interaction have implicated the stroma as more than a mere reactive process or facilitator of tumorigenesis (52) and many current theories support the stroma as an active inducer of tumor formation and

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progression (2, 53). The activated stroma is known to express myofibroblast markers, such as α-smooth muscle actin (α-SMA), and secrete various autocrine and paracrine growth factors, such as transforming growth factor-beta (TGF-β) family members (10, 52).

1.4

New Theories of Cancer Progression: New Pathways

Historically, the theory of pancreatic cancer formation through PanIN lesions was based upon a clonal expansion of a normal somatic cell that had undergone sequential mutations resulting in abnormal behavior. In the last 20 years, the identification of somatic stem or progenitor cell niches has resulted in an explosion of interest in these cells, both for regenerative purposes and for understanding pathologic processes in the human, such as cancer. The theory of pancreatic cancer stem cells and/or stem cells as the cell or origin of pancreatic cancer has gained much enthusiasm and ties together many observations regarding a “dedifferentiated” phenotype, as well as the potential capacity of these cells for infinite renewal of a population of phenotypically heterogeneous cells over a long period of time. This theory also potentially addresses the property of cancer precursor or stem cell chemoresistance (54–56), as the behavior and susceptibility of this cell population may be different than that of the differentiated population of local cells. This ideology regarding the pancreas has been fueled in part by the observed re-expression or mis-expression of various developmental genes during tumorigenesis, which have been known to play a role in maintaining stem cell niches in adults and/or a progenitor cell population in utero. Some of the pathways now implicated in pancreatic cancer include the Notch, Wnt, and Hedgehog pathways, to name just a few (51, 57–71). Their exact roles in pancreatic morphogenesis, morphostasis, and carcinogenesis remain an area of active investigation and an exciting area of research. 1.4.1

Morphogens and the 3D Theory of Cancer

A multitude of different theories of cancer formation have been posited, many of which have focused on the cells of origin of a particular tumor and the various mutations, alterations, and histologic changes these cells undergo. More recently, the emphasis in the field of cancer research has shifted to a focus on the broader milieu in which tumor formation occurs. Much of this literature has been borrowed from developmental biology, and more recently from the stem cell field, in which the stem cell niche and the morphogenic gradients involved in embryologic development have found new popularity. These models integrate the local cellular environment and intracellular pathways active within a given cell into the larger context of the local environment, tissue architecture, and tissue geography as they influence cellular behavior.

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Morphogens are secreted proteins, described initially in the developing embryo, that are capable of eliciting qualitatively different responses from a target cell depending upon concentration. In this case, the distance and location of the target cell in relationship to the source of the morphogen dictates cellular behavior and subsequent tissue patterning. Interestingly, not only do these morphogens therefore activate or suppress various signaling pathways; their distribution actually precedes and dictates the final tissue pattern. Morphogenesis is the process by which patterned embryonic tissue acquires its final form as a result of directional purposeful movement and targeted differentiation. In contrast, morphostasis is a term coined to describe a process similar to morphogenesis, but one that maintains a previously established tissue template. These concepts have been very nicely reviewed and summarized in an article by Potter (72). This process of morphostasis is applicable to the situation in terminally differentiated adult tissues, where the goal is not to create form but to maintain the established microarchitecture (73, 74). In this way, morphostasis is not controlled by a genetic program active within each cell, but rather is a dynamic process involving a cell’s interaction with its environment and the active signaling that occurs between different cell types and tissue compartments. The specific physical conditions important in the maintenance of microarchitecture are the rigidity of the stroma, cytoskeletal tension, interaction of adherens junctions, and maintenance of tissue polarity. Any disruption in these processes can result in a disorder in the local tissue architecture, altering the delicate balance of signals and potentially allowing disordered growth or loss of architecture and therefore, loss of tight cellular control. Of interest, the description of dysregulated morphostasis is quite similar to the well-documented phenomenon of epithelial–mesenchymal transitions (EMT), described at length both in development and in tumor metastasis and invasion (75–77). EMT is a coordinated process occurring in polarized epithelial cells organized in layers in which they become fibroblastoid cells capable of locomotion. During cancer formation and progression, EMT has been associated with tumor invasiveness and metastasis, and has been discussed as a late-stage change in more aggressive or more advanced cancers. Temporally, this process involves the disassembly of cell junctions, cytoskeletal reorganization, loss of epithelial polarity, and remodeling of the cell-matrix adhesions. Implicated in this process of altered morphostasis or EMT are various growth factors and their receptors known to play important roles in cancer, and more specifically, pancreatic cancer development and progression, such as TGF-β, EGF, PDGF, Wnts, MAPK, and various other signaling molecules important in cross-talk between the epithelium and the stroma (75–83). Therefore, the well-known process of EMT may in fact be an alternative description of the process of disrupted morphostasis, with merely a different name ascribed to this process. Regardless of this, it has been overtly clear for a long period of time that one of the most prominent features of cancer is the disruption of normal tissue architecture. Therefore, one of the key initial steps in understanding the development of pancreatic adenocarcinoma is clarifying the native tissue microarchitecture and changes that accompany tumorigenesis (Fig. 11.2).

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Fig. 11.2 Pancreatic microarchitecture. The pancreas is a glandular organ composed of both endocrine and exocrine components. Of interest to the study of pancreatic adenocarcinoma is the microarchitecture of the distal tubular system. The pancreatic ducts and ductules are lined by epithelial ductal cells. The terminal exocrine structures, the acini, are lined by acinar cells, which are cuboidal cells surrounded by fibroblastic-like cells called pancreatic stellate cells. At the junction of the ductal system and the acini are centroacinar cells, which have been implicated in various processes during tissue injury, regeneration, and oncogenesis

1.5 Implicated Signaling Pathways in Pancreatic Adenocarcinoma 1.5.1

K-ras

The transforming gene from Kirsten rat sarcoma virus (K-ras) (Fig. 11.3) was identified more than 20 years ago. Activating mutations of K-ras constitute one of the most frequently activated oncogenes, with 25% of all human tumors harboring these mutations. In the pancreas, activating mutations in K-ras are regarded as the molecular signature of pancreatic cancer, since nearly 100% of tumors have this feature (84), making K-ras one of the most thoroughly studied topics in the field of pancreatic adenocarcinoma research. K-ras is a 21-kDa intracellular, membranebound protein, which is a member of the small GTPase superfamily of proteins. K-ras undergoes a multistage post-translational modification with farnesyl, methyl, and palmityl groups in order for it to be membrane-associated, which is vital for its function (85). K-ras is known to mediate a multitude of cellular activities, including cellular proliferation, survival, and migration (86). Many growth factor receptors utilize K-ras to transduce their downstream signals, and it is a converging point of many disparate signaling pathways. Ras proteins are used to transmit signals from the extracellular to the intracellular environment.

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Fig. 11.3 Activation of K-ras protein. K-ras is activated by the binding of various growth factors to membrane-bound receptors and the recruitment of GRB2 and SOS concurrent with the transition of Ras-GDP to the Ras-GTP activated protein form. Under normal conditions, RAS GTPases (GAP proteins) promote GTP hydrolysis and reversal of the Ras activation step. During oncogenic transformation of Ras, the protein is mutated such that it is rendered constitutively activated, incapable of deactivation by the activity of the GAP proteins

Receptor activation of K-ras occurs via the recruitment of the adaptor protein (GRB2), which subsequently recruits guanine exchange factors (SOS1 and CDC25) to the cell membrane. This enables the membrane to interact with K-ras, resulting in the exchange of GDP to GTP, which forms the activated form of K-ras (K-ras-GTP). Normally this pathway is negatively regulated by GAPs (GTPase activating proteins) that convert GTP to RAS-GDP (see Fig. 11.3). Mutations in several K-ras codons (e.g., 12, 13, and 61) have been identified that render K-ras constitutively active and resistant to GAP inactivation. Other mutations have been identified but their biologic significance remains unknown (23, 87, 88). However, the most common mutation is a single amino acid substitution in codon 12, which has also been implicated in various malignancies, including pancreatic and lung adenocarcinoma, mucinous adenoma, and colorectal carcinoma (2, 89). In humans K-ras mutations are early events in pancreatic adenocarcinoma formation, identified in PanIN-1A lesions (90). However, the mutations in K-ras are identified with increasing frequency with disease progression and are present in nearly 100% of pancreatic adenocarcinomas (25). The current theory states that K-ras functions principally as an initiator of pancreatic cancer. Mutation in K-ras in multiple mouse models has demonstrated that these mutations are sufficient to cause the formation of lesions that resemble human PanINs. The mouse strain K-

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rasG12D was developed to study the role of K-ras using a conditionally activatable K-ras allele in these mice. The alleles are silent in the majority of cells but can be heritably activated by Cre-loxP recombination. The promoter most commonly driving expression Cre expression is Pdx1, which is expressed early in pancreatic development in pancreatic precursor cells. Therefore, all cells within the pancreas will harbor this K-ras mutation. The findings from these studies suggest that activation of K-ras plays a role in pancreatic adenocarcinoma initiation, since these mice develop lesions that closely resemble human PanINs. Of interest, K-ras overexpression does not induce the formation of various other pancreatic tumors, implying specificity of this mutation for pancreatic adenocarcinomas. However, only a small subset of mice went on to develop invasive adenocarcinoma. This implies that K-ras is an initiator, but also that it may need other mutations in order for these lesions to progress to pancreatic cancer. K-ras activation in combination with loss of p16 resulted in a higher percentage and shorter latency to the development of pancreatic cancers, with lesions that also demonstrate increased invasiveness and aggressiveness (24, 91). Although the majority of existing data suggest that K-ras plays its principal role as an initiator, mutations in K-ras may also play an important role in maintenance of the tumor phenotype and for tumor progression (92). Inhibition of K-ras signaling in various tumor cell lines abrogates malignant transformation, suggesting an ongoing need for K-ras signals during cancer progression. One study using immortalized human pancreatic ductal-derived cells was able to demonstrate that activation of K-ras resulted in a loss of density-dependent growth, increasing anchorage independence, and the ability to invade through basement membrane proteins. These studies also showed that these effects were abrogated in the context of MAPK and PI3K inhibition (93). Thus present evidence supports K-ras as a cornerstone of initiating events in pancreatic cancer; however, its in vivo importance as a maintenance factor remains to be determined. Despite the depth of research into the role of K-ras in cancer formation, the precise mechanism by which oncogenic K-ras contributes to initiation and maintenance of the tumors remains to be determined. K-ras not only has numerous growth factor activators, but it also affects multiple signaling pathways. Oncogenic K-ras propagates its signal through at least three primary Ras binding effectors: (1) the phosphoinositide-3-kinase (PI3K) pathway, which is thought to promote cell survival; (2) the RAF-MAPK pathways, which comprise a family of Raf serine/ threonine kinases that activate mitogen-activated protein kinases (MAPKs) and play a key role in cellular proliferation; and (3) the Ral guanine nucleotide exchange factor pathway (RalGEFs), which activates the RalA and RalB Rasrelated small GTPases including the Ras-binding family member Ral guanine nucleotide dissociation stimulator (RalGDS) (93–95). The impact of these pathways on K-ras signaling has been studied using various chemical inhibitors of these downstream cascades, particularly MAPK and PI3K pathway inhibitors. Since mutations in K-ras have been identified in various cancers, there has been much interest in RAS inhibitors with an ongoing debate as to the importance of each downstream signaling pathway in specifically mediating K-ras signaling.

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Ras Effectors RAF-MAP Kinase

One of the most thoroughly characterized Ras effector pathways is that mediated by the mitogen-activated protein kinases (MAPK), also referred to as the extracellular signal-regulated protein kinases (ERKs). These are a group of intracellular protein serine threonine kinases that are known to play important roles in the downstream mediation of various signaling pathways. The MAP kinase pathway consists of three protein kinases. RAF, aka MAPKK kinase or MEKK, phosphorylates and activates a MAPK-kinase (or MEK), which in turn phosphorylates and activates MAPK/ERK. ERK will then activate downstream transcriptional factors to induce transcription of key genes for proliferation (Fig. 11.4) (96). Since these enzymes are integral in such a wide variety of processes with many diffuse effects, it is straightforward to observe an effect in vitro via modulation of the MAPK signaling pathway. However, as this family of signaling molecules is a convergence point for many different signaling cascades, it has also been notoriously difficult to utilize that information for therapeutic interventions. In the setting of

Fig. 11.4 Ras signaling cascades. Signaling via Ras results in the activation of various downstream pathways RAF/MAPK, RalGDS, and P13K. The downstream RAF/MAPK pathway activates MEK, which then acts to activate either JNK or ERK, resulting in the final output of target gene transcription, particularly affecting cellular proliferation. Alternatively, Ras signaling can be mediated by the RalGDS pathway, with eventually intersecting with the cellular polarity signaling pathways, including Rac. Finally, Ras signaling is often mediated by the PI3K pathway, with resultant activation of the AKT/PKB pathway; this inhibits apoptosis or the Rac and/or IKK pathway, which promotes expression of anti-apoptotic proteins such as NF-κB

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pancreatic adenocarcinoma, the RAF-MAP kinase pathway is believed to be critical for the progression and maintenance of tumorigenicity. In vitro studies utilizing dominant negative MEKK demonstrate an inhibition of pancreatic cancer cells’ growth with abrogation of MEKK signals (97). Inhibition of RAF/MAPK not only results in an inhibition of K-ras–mediated growth, but also facilitates tissue invasion (93). In vivo, pharmacologic inhibition of kinase suppressor of ras-1, a transducer of the Ras-Raf signaling pathway, is able to inhibit K-ras-mediated pancreatic cancer formation in murine xenografts (98, 99).

1.6.2

PI3 Kinase-AKT-PTEN

Phosphatidylinositol-3-kinases (PI3K) constitute a large and complex family of lipid kinases. They are implicated in several different cellular functions, including proliferation, differentiation, chemotaxis, survival, cellular trafficking, and glucose homeostasis (100). There are three classes of these proteins with multiple subunits and isoforms. Class IA PI3Ks are heterodimeric proteins composed of a p110 catalytic subunit and a p85 regulatory subunit. These proteins are activated through interaction with the phosphotyrosine residues of various receptor tyrosine kinases or through binding of active K-ras to the p110 catalytic subunit. The downstream signals are mediated through the action of the survival signaling kinase AKT/PKB. AKT/PKB plays a significant role in the regulation of apoptosis through proapoptotic proteins such as Bad and caspase 9 (101). There are three known sites of mutations that elevate the lipid kinase activity of PIK3CA (a catalytic subunit of PI3K) and activate the AKT signaling pathway (102), and many studies have reported high frequencies of somatic mutations in the PIK3CA gene in various human solid tumors (103). Studies looking specifically at pancreatic cancer cell lines have demonstrated that PI3K signaling is necessary for growth and survival of tumor cells (104), and as a major effector of K-ras signaling, PI3K is well situated to play a role in pancreatic adenocarcinoma. Mice have been engineered in which the endogenous PI3K gene has a mutant p110α subunit, which is enzymatically active but incapable of binding to and interacting with K-ras. Therefore, activation of K-ras signals cannot be transmitted through the PI3K network. In this context, mouse embryonic fibroblasts were treated with various growth factors, including EGF and FGF2, demonstrating an inability of these cells to now respond to growth factor signaling with blockade of PI3K activation. In addition, this system was then applied to several well-established models of cancer formation demonstrating an overt inhibition of oncogenesis with blockade of downstream PI3K signals. These findings suggest that the interaction of K-ras with the PI3K subunit p110α is required for the development and progression of malignancies, likely mediated by various extracellular signaling proteins (105, 106). An alternative target of the PI3K pathway is Rac, a G protein that can be activated by phosphatidyl 3,4,5-triphosphate (PIP3) (107). Rac is important in regulating anti-apoptotic gene expression through NF-κB, and cytoskeletal rearrangements

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(108). Ras-controlled membrane remodeling through Rac is dependent on PI3K activity (109). Thus, as we begin to know more about affectors and effectors on the PI3K pathway in pancreatic cancer, it appears that PI3K is an important mediator of Ras-induced cell transformation. As the models in which many of these studies are done are murine, the caveat is the unknown relevance these systems may have to the actual situation of humans in vivo (106).

1.6.3

AKT/Protein Kinase B (PKB) and PTEN

AKT, also known as protein kinase B (PKB), is a serine-threonine protein kinase known to be a key downstream mediator of the aforementioned PI3K pathway (110), and inhibition of AKT via chemical suppression or transgene expression has a clear impact on tumorigenesis, with resultant alterations in cell growth and survival (110, 111). AKT is capable of suppression of apoptosis in a transcriptionindependent manner through direct phosphorylation and inactivation of the apoptotic machinery (112). The AKT gene exists in multiple alternatively spliced variants. Of interest to pancreatic cancer research is the fact that this gene is amplified and/or constitutively activated in up to 60% of pancreatic cancers (111, 113). Recently the interaction of the AKT inhibitor PTEN has also been examined in the context of pancreatic adenocarcinoma (51). PTEN is an interesting protein, identified as a tumor suppressor and found mutated in multiple different cancers at high frequency (110). Functionally, it is a protein tyrosine phosphatase, but unlike most of these phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It is therefore a negative regulator of various proteins and functions as a tumor suppressor by inhibiting the AKT/PKB signaling pathway (114). It has been observed that PTEN point mutations are rare in pancreatic cancer; however, functional inactivation occurs with high frequency. This inactivation occurs through multiple different mechanisms including promoter methylation, inhibition of protein synthesis, and/or mRNA expression (115, 116). In order to elucidate the potential role of PTEN in pancreatic cancers, a mouse model with pancreas-specific deletion of the PTEN gene has been created. In these mice with PTEN deletions (and therefore implied activation of AKT signaling) there are progressive pancreatic abnormalities throughout development and maturation, with the development of diffuse ductal mucinous metaplasia as well as the evolution of PanIN lesions with occasional malignant transformation. As PTEN is an inhibitor of PI3K signaling through AKT/PKB, the studies suggest that there may be an association between PI3K signaling and pancreatic cancer (51). That said, PTEN has other functions that are independent of its activity as a PI3K antagonist (e.g., p53 modulation) (117), and it is unclear to what extent, if any, these factors influence the formation of pancreatic lesions. Other downstream effectors of AKT, such as mammalian target of rapamycin (mTOR), have recently been under investigation as mediators of tumorigenesis.

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In pancreatic ductal adenocarcinoma, 55–65% of tumors have an activated AKTmTOR pathway. mTOR inhibitors can reduce malignant progression in murine xenograft models of pancreatic cancer and in lung cancer induced by mutant K-ras (118, 119).

1.7

RalGEF/RalGDS Pathway

The RalGEF family contains at least three members containing Ras-binding domains, including RalGDS, Rgl, and Rgl2/Rlf. RalGEF’s role is to regulate the function of the G proteins, RalA and RalB, in the same manner that SOS and CDC25 regulate Ras activity, i.e., by stimulating the exchange of GDP for GTP (120, 121). RalGDS is a key component of Ras-dependent carcinogenesis in vivo; this is supported by the observation that absence of RalGDS in vivo results in reduced tumor incidence, size, and progression to malignancy in multistage carcinogenesis of the skin, as well as reduced transformation by Ras in tissue culture. In these systems RalGDS does not appear to participate in the regulation of cellular proliferation, but instead appears to control the survival of transformed cells (122). In humans RalGEFs have been shown to be potent transforming factors (123), and knockdown of RalA expression impedes, if not abolishes, the ability of human cancer cells to form tumors. RalA has also been noted to be activated in a panel of cell lines from pancreatic cancers (124), suggesting that activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

1.8

K-ras Cross-Talk

K-ras may interact with known morphogens to exert its effects on the initiation of pancreatic cancer. Other interesting work has postulated that oncogenic K-ras may promote pancreatic tumorigenesis in part through activation of the Hedgehog (HH) pathway. In models using pancreatic ductal adenocarcinoma (PDA) cells, it has been observed that K-ras activation is capable of activating and maintaining Hedgehog signaling (see the following). Hedgehog proteins are extracellular secreted growth factors known to play important roles in patterning during embryogenesis. More recently Hedgehog proteins have been implicated in pancreatic adenocarcinoma progression through the MAPK pathway. Gli proteins are DNA-binding transcription factors that mediate sonic Hedgehog (Shh) signaling. In the setting of K-ras activation there is activation of Gli protein expression as well as inhibition of the degradation of Gli2 protein. This allows increasing levels of Gli1 protein to accumulate and presumably participate in intracellular signaling in cells with persistent K-ras activation (125). These observations suggest a role for Gli activation by oncogenic K-ras during transformation of human pancreatic ductal cells in vitro.

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Of relevance to these studies is the observation that overexpression of Gli1, along with other HH signaling molecules such as Shh, Ihh, Smo, and Patched, is found in human pancreatic cancer tissues and pancreatic cancer cell lines (67). Furthermore, the administration of a MEK-specific inhibitor results in the suppression of K-ras– mediated Hedgehog activation via Gli1 (125). In addition, expression of a constitutively active form of BRAF was able to overcome this upstream blockade of K-ras signaling, again implying a role for MAPK in mediating the activation of Hedgehog signaling by oncogenic K-ras. PTEN-AKT signaling has also been examined in other model systems of cancer formation and progression, such as the intestine, with some interesting results. Targeted gene deletion of PTEN in the mouse intestine has been demonstrated to enhance the formation of precancerous polyps composed of crypt-like tissue and stroma. These effects are believed to be mediated by PTEN-AKT modulation of the Wnt/β-catenin pathway, resulting in β-catenin nuclear localization and activation of the downstream Wnt pathway (126). Wnt proteins are secreted glycoproteins known to play important roles in the maintenance of stem cell compartments and in oncogenesis, as well as to play various roles in patterning and cellular behavior during development (127). The cross-talk between these two divergent signaling pathways is intriguing, as both pathways have been implicated in pancreatic tumorigenesis.

1.9

Affector Pathways

There is a wide range of stimuli that can initiate Ras signaling. The complexity of Ras activation results from a vast array of upstream receptors, including receptor tyrosine kinases, integrins, heterotrimeric G-proteins, and cytokine receptors (128– 130). However, the best known and best-described pathway is stimulation of K-ras via a receptor tyrosine kinase such as the EGF receptor (Fig. 11.5).

1.10

Receptor Tyrosine Kinase (RTK)

The EGFR family consists of four transmembrane tyrosine kinases: EGFR (erbB-1, human EGFR; HER); erbB-2 (HER-2); erbB-3 (HER-3); and erbB-4 (HER-4) (131). These receptors are all composed of an extracellular domain, a hydrophobic transmembrane domain, and a tyrosine kinase intracellular cytoplasmic domain. The classical EGF receptor (EGFR) is known as Her1 or ErbB1 (89). The mechanism of action of this receptor begins with the binding of a ligand to the EGF receptor, which induces oligomerization of the receptor, which allows the juxtaposition of cytoplasmic and catalytic domains enabling activation of the kinase activity and resultant transphosphorylation of the receptor (132, 133). Although EGF is considered an inducer and upstream activator of K-ras signaling,

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R FEG

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Fig. 11.5 EGF and EGF-related signaling. The epidermal growth factor (EGF) family of receptor tyrosine kinases is composed of various receptors, including the Her2Neu orphan receptor and the EGF-related ligands EGF, TGF-α, amphiregulin, HB-ECF, β-cellulin, neu-differentiation factor, and heregulin, which exist as membrane-bound proteins prior to activation and initiation of signaling. The EGF receptor proteins contain an extracellular ligand-binding region, a transmembrane segment, and a cytoplasmic tyrosine kinase domain. These proteins function as dimers, either heterodimeric or homodimeric complexes, that are capable of activating various downstream pathways including the RAF/MAPK and P13K pathways. EGF-R dimerization results in the recruitment of various Src homology 2 (SH2)–containing effector proteins to the membranebound receptor complex. Upstream of the Ras pathway, the EGF receptor signal activates the GRB2/SOS complex, resulting in Ras activation (as seen previously), with subsequent activation of the PI3K and MAPK pathways

there is also a positive feedback loop in which activation of K-ras actually stimulates the production of EGF receptors, allowing increased responsiveness of the cells to the incoming signals. Pancreatic ductal adenocarcinoma cells (PDACs) express high levels of EGFR, HER-2, HER-3, and, to a more limited extent, HER-4 (134, 135). PDACs also express high levels of the EGF family of ligands (see the following) (136). EGFs and EGFRs are able to activate autocrine signaling cascades involving PI3K. This activation of EGF signaling is observed in even the earliest PanIN lesions, suggesting that these signals may be active in initiating pancreatic cancer. However, more important than the temporal expression of these proteins in pancreatic lesions is the finding that pancreatic adenocarcinomas undergo growth inhibition in the context of suppression of EGF signaling by blocking antibodies or by dominant negative expression constructs (2), thereby demonstrating functional consequences of EGF signal modulation in tumorigenesis. Pancreatic cancers that overexpress ERBB3 (HER3) are known to be sensitive to inhibition by Erlotinib (Tarceva) (137). Presently there are clinical

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trials looking at the efficacy of this drug in humans with pancreatic cancer based upon the favorable in vitro studies. EGFR-related peptide (ERRP), a recently isolated pan-erbB inhibitor that targets multiple members of the EGFR family, seems to attenuate EGFR activation (138). ERRP applied to pancreatic adenocarcinoma studies causes marked inhibition of pancreatic cancer cell growth. These changes are accompanied by increased apoptosis and a concomitant attenuation of Notch-1 and NF-κB expression and down-regulation of NF- κB downstream genes, such as matrix metalloproteinase-9 and vascular endothelial growth factor. These changes result in an inhibition of pancreatic cancer cell invasion, as assayed by transit through Matrigel (139). Activation of the EGFR pathway is also likely to contribute to the maintenance of tumors by promoting neovascularization through stimulation of vascular endothelial growth factor (VEGF) expression through the JAK/STAT pathway (140).

1.10.1

Her-2/neu/ErbB2

Her-2/neu, also known as ErbB2, is a 185 kDa glycoprotein receptor, structurally homologous to the epidermal growth factor (EGF) receptor. There are multiple different transcriptional variants, although the distinctions between these proteins remain to be fully characterized. Of note, Her-2/neu is called an “orphan” receptor due to the fact that, although it bears structural similarity to EGF receptor (EGF-R), it is not capable of binding to EGF ligands. Instead, it is capable of dimerization with EGF-R to form a heterodimer. This interaction stabilizes EGF-R ligand binding and enhances kinase-mediated activation of various downstream signaling pathways, such as the MAPK and PI3K pathways. Amplification and/or overexpression of Her-2/neu have been associated with various cancers, including breast, ovarian, and, of relevance to our studies, pancreatic malignancies. There have been conflicting reports in regard to the importance of this pathway in pancreatic cancer. It was recently demonstrated by FISH analysis that in pancreatic adenocarcinoma 25% of tumors have HER2/neu gene amplification (141). Herceptin, a monoclonal antibody to Her2/neu, has been shown to suppress tumor growth both in vivo and in vitro in pancreatic cancer cell lines that overexpress HER-2/neu (142). There are also data to suggest that Her-2/neu expression correlates with shorter survival times in patients with pancreatic cancer (89), although its predictive or prognostic role remains unclear.

1.10.2

EGFR Ligands

Epidermal growth factor (EGF) is a potent mitogen in a variety of cells, with profound effects on the differentiation of specific cells in vivo and in vitro, including cells of both epidermal and mesenchymal lineages. The EGF precursor protein exists as a membrane-bound molecule, which is proteolytically cleaved upon activation to generate a peptide hormone that stimulates cells to divide. Members of the

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EGF family include EGF itself, TGF-α, amphiregulin, HB-ECF, β-cellulin, neudifferentiation factor, and heregulin. Pancreatic cell lines are recognized to produce large amounts of TGF-α and TGF-β, IGF-1, and the β chain of platelet-derived growth factor (PDGF). TGF-α and IGF-1 are thought to stimulate pancreatic cell growth in vitro through a postulated autocrine mechanism. The role of TGF-α is believed to be as an initiator and trophic factor in pancreatic cancer. In genetically engineered mice, TGF-α overexpression in the pancreas causes the development of tubular structures and fibrosis, and mice older than 1 year develop ductal pancreatic cancer. Interestingly, crossbreeding these mice with p53 knock-out mice dramatically accelerates tumor development. Moreover, tumors developing in these mice show frequent bi-allelic deletion of the Ink4a locus or LOH of SMAD4 (143, 144). Notch activation is required for TGFα-induced changes in epithelial differentiation (58), again demonstrating cross-talk between various key signaling pathways during oncogenesis. Aberrant regulation of the IGF-1 autocrine loop is also associated with increased pancreatic tumorigenicity. This effect seems to be potentiated by the loss of response to TGF-β in tumor cells lacking the TGF-β receptor type RII (145).

1.11 Chemotherapeutic Targeting of K-ras and Effector Pathways K-ras signaling regulates the initiation and progression of pancreatic cancer through multiple pathways capable of altering proliferation, apoptosis, angiogenesis, and migration, thereby making it a logical target for cancer therapy. Approaches targeting K-ras for cancer treatment have included inhibition of Ras protein expression by antisense oligonucleotides, as well as RNA interference. Unfortunately, the antisense approach has been limited and it has not been convincingly demonstrated to have any anti-tumor activity in human trials (146). The second approach is to use double-stranded RNA to interfere with and reduce the expression of oncogenic Kras. In vitro these studies show some positive effects (147); however, K-ras-targeted siRNA have yet to enter clinical trials. Therapeutic targets thus far have included the inhibition of K-ras processing through farnesyltransferase inhibitors (FTIs) or a combination of farnesyltransferase and geranyltransferase inhibitors (FTI/GGTI). FTI have been used successfully for therapy of AML, but as yet have not been shown to have significant antitumor activity in pancreatic cancer studies (148). Attempted therapeutic strategies have also included targeting of the mutant K-ras protein via immunologic therapy or peptide inhibitors. These approaches using Ras peptide-based antibodies have shown some promise in the treatment of pancreatic cancer (149), but future studies are need to clarify an exact role in pancreatic cancer therapy. Most recently, many new inhibitors have been developed to target Ras effectors, such as Raf kinase, MEK, and mTOR. Raf inhibitors such as Sorafenib (BAY 43-9006) not only target the Raf/MEK/Erk signaling pathway, but also have demonstrated effects on the VEGFR and PDGFR pathways. Unfortunately, although Sorafenib appears to

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have efficacy in renal cell cancer in various clinical studies, there has been no such benefit demonstrated in patients with pancreatic cancer (150). Another strategy utilizes the MEK inhibitor, CI-1040, an oral selective small-molecule inhibitor of MEK 1-2. Unfortunately, a phase II study in 67 patients with four different tumor types including pancreatic cancer found no responses to treatment with CI-1040, although the treatments were well tolerated (151). Several mTOR inhibitors are currently being tested in clinical trials. Phase 1-3 studies have demonstrated some encouraging results in a variety of human tumors; however, their role as potential treatments for pancreatic cancer is still under investigation in a Phase II study using RAD001 in patients with metastatic pancreatic cancer.

1.12

Mesenchymal Pathways

1.12.1

TGF-β

TGF-β (Fig. 11.6) is a multifunctional protein with established roles in proliferation, differentiation, and various other cellular activities (11, 152, 153). In many systems it has been established that TGF-β can act synergistically with TGF-α to induce transformation (154-156). However, in other settings TGF-β alone may act as a negative autocrine and/or paracrine growth factor, and dysregulation of TGF-β activation has been associated with apoptosis (157). Many divergent cell types express TGF-β and almost all of them also have receptors for this protein (81, 152, 153, 158). The different members of the TGF-β family all function through the same receptor signaling system. In the setting of oncogenesis, TGF-β can function as both a tumor suppressor and a promoter of tumorigenesis. In the first context, as a tumor suppressor, TGF-β inhibits proliferation of cells and is also capable of inducing apoptosis (159). Of relevance to the field of pancreatic cancer research, TGF-β can act as a growth inhibitor of epithelial cells and can inhibit epithelial cancer initiation in vivo (160). For example, increased TGF-β signaling has been noted to suppress tumor formation in transgenic mice (152). Conversely, loss of TGF-β signaling has been associated with carcinogenesis. In humans, inactivating mutations of TGF-βrelated signaling proteins (e.g., Smad4) have been found in many cancers, including pancreatic tumors. Interestingly, loss of TGF-β sensitivity of tumor cells is often accompanied by increased expression of TGF-β by these same cells. In addition, elevated levels of plasma TGF-β levels are seen in many of these patients with cancer and may be a predictor of early metastasis (161). There are several potential explanations for this phenomenon. On the one hand, TGF-β may be activating different downstream signaling pathways in different temporal contexts or at different levels of expression. Alternatively, the high levels of TGF-β may be reactive to the insensitivity of the system to TGF-β (162). Many types of advanced carcinomas become resistant to the TGF-β–mediated growth inhibition, and the theory posits that the role of TGF-β in these contexts switches from that of growth inhibition to tumor promoter by enhancing invasiveness and metastatic potential.

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Fig. 11.6 TGF-β signaling. TGF-β proteins bind to a type II receptor dimer, which then recruits a type I receptor dimer to the membrane, resulting in a tetrameric complex of membrane-bound serine/threonine kinase receptors. Receptor-bound Smad 2 is phosphorylated and activated, resulting in an intracellular cascade leading to Smad4 activation and translocation to the nucleus. This initial phosphorylation step can be blocked by the inhibitory Smad, Smad7. Downstream TGF-β signaling is mediated by a combination of Smad2/3 and Smad4 activation. The phosphorylated Smad2/3 and Smad4 complex translocates to the nucleus and activates transcription of various downstream target genes important in cellular proliferation as well as angiogenesis and apoptosis. TGF-β is also capable of activating various non-Smad–dependent pathways as well, including the Rho/ROCK cellular polarity pathway and MAPK pathways

In the setting of pancreatic adenocarcinoma, there is often the expression of three different TGF-β isoforms as well as altered expression of the various TGF-β receptors (33, 39, 44, 159). Inactivating mutations of the TGF-β receptors (types I and II) have also been noted in specimens from pancreatic adenocarcinomatous lesions. Similar to observations in other systems, pancreatic cancer cells are often resistant to the growth-inhibitory effects of TGF-β, and this correlates with Smad4 mutations that have been observed in these cancer cells (43, 153, 153a). As mentioned, mutations in Smad4 appear to play an integral role in pancreatic carcinogenesis, as demonstrated by the observation that 55% of pancreatic tumors have chromosomal deletions and inactivating mutations in Smad4 (153a). Smad4 is a downstream mediator of TGF-β signaling and is implicated in the growth inhibition and anti-angiogenic properties of TGF-β (164). TGF-β inhibits the growth of most epithelial types either by blocking the G1-S cell cycle transition or by inducing apoptosis. These cellular responses are partially, but not exclusively, Smad4dependent (152, 153). These alterations in cellular behavior can be enhanced by activated oncogenes, such as K-ras, which cooperates with TGF-β signaling during

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cellular invasion. In vivo observations have correlated disruption of the TGF-β signaling cascade via Smad7, a TGF-β downstream inhibitor, with premalignant ductal lesions of the pancreas (139). Observations of Smad4 mutations in pancreatic cancer suggest that these mutations occur later in cancer progression, such as at the stage of PanIN3 lesions and adenocarcinomas, as these mutations have not been associated with flat or papillary intraductal neoplasias (43, 153, 153a). It is believed that alterations in Smad4 expression may be a molecular prognostic marker for pancreatic cancer. There is increased expression of mutated versions of Smad4 in poorly differentiated versus well-differentiated cancers, and studies of patient outcomes have demonstrated that patients without mutations do better after tumor resection than those with Smad4 mutations (42, 164a). Speaking to the importance of modulation of the TGF-β signaling cascade in pancreatic cancer is the observation that mutations in Smad4 are a common genetic alteration identified in pancreatic cancer, while they occur with a low incidence in other cancers (89).

1.12.2

Smad4/DPC4

Smad4 is a 64-kDa protein known to be important in gene transcription and growth arrest. During TGF-β–mediated signaling, a downstream cascade results in protein phosphorylation and nuclear translocation of Smad4 in complex with other intracellular proteins (152, 153, 160). In the context of pancreatic cancer, the high incidence of loss-of-function mutations in Smad4 and various other disturbances in the TGF-β cascade may result in a loss of tumor suppression and the acquisition of a more proliferative and invasive phenotype. These tumors then become resistant to the antiproliferative effects of TGF-β (153, 164b). It remains unclear whether alterations in Smad4 exert their primary effect on the epithelial tissue itself or instead modulate tumor formation through alterations in the tumor-associated stroma. Several studies suggest that the latter might be the case (164, 165, 166). Interestingly, re-expression of Smad4 in human colon and pancreatic cancer cells suppresses tumor cell growth in vivo but not in vitro, suggesting that changes in the local tissue environment may contribute to the Smad4-mediated events, which can be seen more readily in the context of the dynamic signaling present in vivo rather than in an in vitro system. Recent studies have examined the role of Smad4 during pancreatic development with, surprisingly, no phenotypic changes in pancreatic tissue development in the absence of Smad4. However, when loss of Smad4 occurs in combination with activation of Kras, pancreatic neoplasms rapidly form. Isolated expression of K-ras in this system results in PanIN lesions with very slow turnover to malignancy. Yet in K-ras mutant mice with Smad4 deficiencies, tumors rapidly develop (153a). There are several clinical syndromes that have been associated with Smad4 mutations. In juvenile polyposis syndrome (JPS), patients demonstrate an overgrowth of native-type tissue in the intestine, resulting in these multiple polyps. Of note, the loss of normal genetic function in this syndrome occurs predominantly in the interstitial fibroblasts among the colonic polyps. Interestingly, there does not

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appear to be a predisposition in the patients to pancreatic adenocarcinoma, suggesting that other contributing mutations and factors must be present in the pancreas for cancer formation to occur. In the pancreas, loss of Smad4 function is a predictor of decreased patient survival, consistent with a role in disease progression rather than disease initiation (2). Interestingly, loss of Smad4 expression may not be the key mediator of the loss of TGF-β responsiveness seen in many pancreatic tumors. This is suggested by observations that responsiveness of tumor cells is not re-established with forced expression of Smad4 in these tumors and the observation that in vitro responsiveness in various cell lines does not correlate with Smad4 loss. One mechanism proposed for this loss of TGF-β sensitivity instead is the loss of the TGF-β1 receptor. In various in vitro and in vitro models, blockade of TGF-β to TGF-β receptor signaling (either via soluble receptor or blocking Ab) results in decreased oncogenesis and tumor invasiveness (167, 168).

1.12.3

Hepatocyte Growth Factor

Hepatocyte growth factor (HGF) (Fig. 11.7) also known as scatter factor, is a secreted protein known to play important roles in cell growth, motility, and morphogenesis and is structurally homologous to various enzymes active in the coagulation cascade. HGF signals in an active heterodimeric form by activation of the transmembrane cell surface proto-oncogenic c-Met receptor (169–174). HGF is secreted by mesenchymal cells and acts as a paracrine cytokine on cells of mainly epithelial origin. Therefore, HGF is primarily expressed by fibroblasts, whereas the HGF receptor, c-Met, is expressed primarily by epithelial tissues. HGF is important in various processes such as angiogenesis, tumorigenesis, branching morphogenesis, and tissue regeneration, as it is a very potent mitogen, exerting effects on cellular motility as well as facilitating the ability to invade a given matrix (169). From a protein standpoint, HGF is secreted as an inactive polypeptide, which is cleaved into a 69-kDa α chain and a 34-kDa β chain. The active heterodimeric protein is produced by combination of the α and β subunits via a disulfide bond. Interestingly, alternative splicing of this gene can lead to many different transcript variants, which produce multiple different HGF isoforms. The known role of HGF in paracrine signaling from the mesenchyme to the epithelium makes HGF an optimal candidate as a mediator of stromal–tumor interaction in the setting of pancreatic adenocarcinoma. Implicating HGF signaling in the development of pancreatic cancer is the observation that there is increased expression of both HGF and the c-Met receptor in PanIN lesions and the progression of pancreatic cancer formation (175, 176). The temporal and experimental results examining HGF in pancreatic adenocarcinoma suggest a potential role for HGF signaling during tumor initiation, although increased expression of c-Met is also seen in more advanced PanIN lesions. Studies of HGF function have demonstrated a role in motility of PDACs in vitro, and blockade of HGF signaling results in inhibition of tumor invasion and angiogenesis (35–37, 175–178).

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Fig. 11.7 HGF and PDGF signaling. HGF binds to a dimer of α- and β-subunits of the c-Met receptor. HGF signaling is capable of activating multiple downstream pathways, including the Ras signaling pathway with subsequent MAPK and ERK signaling, the PI3K pathway, as well as proteins involved in cellular cytoskeletal arrangement and motility. PDGF also binds to a dimeric receptor and is also capable of activating both the Ras and PI3K pathways, again emphasizing the many sites for potential cross-talk between these various growth factor pathways

Studies using irradiated pancreatic fibroblasts have demonstrated an elevated metastatic potential of developing tumors when associated with irradiated fibroblasts. These changes are associated with increased expression of c-Met by the carcinoma cells concurrent with increased expression of TGF-β by the irradiated fibroblasts. Of note is the observation that there was no concomitant increase in HGF secretion by these fibroblasts, although this does not exclude the potential role of an HGF-related ligand or ligand-independent activation of the c-Met receptor. Theories linking c-Met signaling and HGF activation with tumor–stromal interactions are strengthened by the observation that specific antagonists of HGF are able to block the enhanced invasiveness of pancreatic carcinoma cells both in vitro and in vitro (178). These types of observations are the foundation for current clinical trials utilizing a soluble c-Met receptor (decoy met) for cancer treatment (162).

1.12.4

Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF) (see Fig. 11.7) is a member of a family of mitogenic factors known to be of mesenchymal origin and structurally characterized by a motif of eight cysteines. PDGF-A and PDGF-B can exist as either

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homodimers or heterodimers depending upon the context, connected via disulfide bonds. PDGF signals through its cognate receptor, PDGFR, which forms a homodimer or heterodimer, which results in receptor autophosphorylation. Studies of PDGF function using knock-out mice have demonstrated cellular effects of these proteins on various cell types. PDGF knock-out mice usually die as embryos or shortly after birth, emphasizing the importance of PDGF in various developmental processes (179). PDGFs and PDGFRs have been implicated in various processes in tumorigenesis, both in the setting of pancreatic cancer as well as other gastrointestinal cancers and other cancer systems. PDGF appears to be involved in growth stimulation of tumor cells, tumor angiogenesis, recruitment of tumor fibroblasts, and the tumor desmoplastic response. Interestingly, PDGF receptors in the tumor stroma have become recent targets of drug therapy as their roles in regulating tumor interstitial fluid pressure, transvascular transport, and tumor drug uptake become more apparent (180, 181). In studies of pancreatic fibroblasts, PDGF has been touted as an effective mitogen, enhancing the migratory capacity of these pancreatic stromal cells (PSCs) and inducing AP-1 binding in these PDGF-activated cells (10). In the context of pancreatic adenocarcinoma, immunohistochemical studies have demonstrated the expression of phosphorylated PDGFR-α or PDGFR-β in both tumor cells and tumor-associated endothelial cells. Comparison of in vivo to in vitro cultures of pancreatic cells demonstrates increased expression of PDGF and PDGFR in in vivo systems. Tumor activity can be functionally inhibited with blockade of PDGF signaling via treatment with the tyrosine kinase inhibitor, STI571 (Gleevec) in combination with gemcitabine, which results in inhibition of tumor growth and metastasis (182).

1.12.5

Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are a family of enzymatic proteins involved in the breakdown of extracellular matrix (ECM) during normal physiologic processes, such as during embryonic development, reproduction, and tissue remodeling, as well as in cancer metastasis. Most MMPs are secreted as inactive pro-proteins, which are activated through cleavage by extracellular proteinases. These MMPs are capable of degrading various protein types, including collagen subtypes. Many murine models also suggest a role for MMPs in tumor-associated tissue remodeling. In keeping with this theory, levels of MMP activity are found in various tumor types, and in well-established models MMP2 and MMP9 knockout mice have a reduced susceptibility for lung metastasis. These effects may be mediated by growth factors activated by MMPs, such as IGFs, FGFs, and TGF family members (162). MMPs as targets for cancer therapy have been the focus of several clinical studies with mixed outcomes in regard to the role of MMP inhibitors (MMPIs) on cancer progression. Several Phase III drug trials of MMPIs were discontinued secondary to reports of no clinical efficacy with MMP inhibition in various cancers. However, more recent studies of the role of MMPs in cancer suggest that MMPs may be more active in tumor–stromal interactions and tumor

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angiogenesis, rather than as effectors on tumor cell behavior directly. This being the case, the role of MMPs in the treatment of cancer may need to be refocused to examine the effect of MMP modulation in earlier stages of cancer or in combination with other therapeutics for a combined effect on tumor cell activity and tumor–stromal interactions (183).

1.13

Developmental Regulators

1.13.1

Wnts

Wnt proteins (Fig. 11.8) are a family of secreted glycoproteins responsible for signaling during many stages of embryologic development. Wnts signal through the membrane-bound receptor family, Frizzleds, often in cooperation with the Wnt co-receptors LRP-5 or LRP-6. Downstream Wnt signals mediate a cascade in which b-catenin in the cytoplasm, which is normally serine/threonine phosphorylated and tagged for ubiquitin proteasome degradation, is protected from this

Fig. 11.8 Canonical Wnt signaling. In the absence of Wnt proteins, intracellular complexes composed of Axin, APC, and GSK-3 are formed which are responsible for the serine/threonine phosphorylation of the cytoplasmic pool of β-catenin. This phosphorylating step tags β-catenin for ubiquitin proteasome-mediated degradation, thus keeping intracellular levels of this protein low. In the presence of canonical Wnt signaling, a receptor complex composed of the Wnt receptors Frizzled, as well as the co-receptor LRP5/6, is formed. This leads to a destabilization of this AxinAPC-GSK3 complex and a resultant protection of β-catenin from this phosphorylation step. Cytoplasmic levels of β-catenin increase with translocation of the protein to the nucleus and activation of downstream signaling mediated by the TCF/LEF family of transcriptional activators

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degradation and eventually translocates to the nucleus to initiate transcription through the TCF/LEF family of transcriptional activators (184). More recently, Wnt signaling has been implicated in the maintenance of various stem cell populations in humans (185), which is a logical extension of the presumed mitogenic role Wnt signaling plays during embryogenesis. Wnts have historically enjoyed a high level of interest as developmental regulators as well as oncogenes, with implication of this signaling pathway in various forms of cancer (186). During development, Wnts play irreplaceable roles in the formation of various organ systems, directing both organ growth and patterning. In the context of tumorigenesis, Wnts have been studied in depth in various systems. In terms of the gastrointestinal system, the role of Wnt signaling has been clarified quite extensively in the intestine, resulting in a clear model of the Wnt activity in intestinal homeostasis and dysregulation of Wnt signaling in colon cancer formation and tumorigenesis. First, active Wnt signaling has been demonstrated to keep intestinal cells dividing and in a state of continued proliferation. Simultaneously, Wnt activation prevents terminal differentiation of cells as they continue in a proliferative and undifferentiated state. Finally, specific to the intestine, Wnt signaling confers the potential (but not the obligation) to become a secretory cell type (e.g., Paneth cell). In contrast to the Paneth cells, all terminally differentiated cells in the intestine demonstrate no maintenance or persistence of Wnt activation outside of the stem cell compartments or those niches in which oncogenic transformation is occurring (187). The combination of these observations clarifies the role of Wnt signaling as a mediator of the intestinal stem cell niche and a key factor in maintenance of an undifferentiated pool of progenitor cells, with release from Wnt control as cells progress toward a differentiated phenotype. Parallel observations have resulted in a large volume of literature describing similar roles for canonical (or β-catenin–mediated) Wnt signaling in various other progenitor populations in the adult system and during oncogenic transformation of various tissue types (127, 185). In the pancreas, Wnt signaling has been implicated in formation of the exocrine pancreas as well as in maintenance of the endocrine pancreas. Expression of Wnts has been noted in both the epithelial and mesenchymal compartments of the pancreas during organogenesis (61). Work by Wells et al. (188) has recently demonstrated a critical role for β-catenin signaling in the formation of the exocrine pancreas, as β-catenin knock-out mice (using the Pdx1 promoter) demonstrate hypoplastic pancreata with a significantly decreased exocrine compartment in the presence of a normal islet cell compartment. These observations have led to the current hypothesis that canonical Wnt signaling may be involved in expansion of a population of exocrine precursors during pancreatic development (188, 189), which is congruent with the purported role of Wnt signaling in various other organ systems. Interestingly, deletion of β-catenin in adult acinar cells has no effect on cellular function, suggesting a more isolated role for β-catenin signaling during development and initial differentiation, and a suggestion of potential reactivation in a subset of cells in the setting of chronic injury and/or oncogenesis. This is remarkably similar to the aforementioned role of canonical Wnt signaling in the intestine and other cancer models.

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Recent work has demonstrated proliferative effects of Wnts on pancreatic β cells both in vitro and in vivo, with associated functional alterations in β cell activity including alterations in blood glucose homeostasis. This suggests a modulatory role of Wnt signaling in the endocrine pancreas (190). In the setting of pancreatic adenocarcinoma, the downstream effector of canonical Wnt signaling, β-catenin, is found localized to the nucleus in various high-grade PanINs and malignant lesions, suggesting re-activation of this signaling system in pancreatic cancer. Nuclear localization of β-catenin correlates with activation of downstream mediators of Wnt signaling, including the LEF/TCF family of transcription factors (191). Tumor specimens have also been examined for expression of Wnts and various Frizzled receptors, with observational reports of increased Wnt1 and Fz2 levels, as well as decreased serine/threonine tagged β-catenin (i.e., that subset of β-catenin targeted for degradation) in 65% of tumor specimens, again suggesting canonical Wnt activation during tumor formation (192). In a mouse model of Wnt activation, the APC min mouse (with inactivation of a suppressor of Wnt signaling), there is a predisposition of the mice to colon polyps, but also to abnormal acinar foci that demonstrate high levels of β-catenin. Upon further study in this system, the combined activation of Wnt signaling combined with inactivation or loss of p53 results in a progression of these abnormal pancreatic foci to adenocarcinoma, suggesting a role for Wnt signaling and cross-talk with other important oncogenic mediators during tumorigenesis. In studies examining the role of Smad4 in pancreatic adenocarcinoma, TGF-β/Wnt interaction has been suggested by the finding of increased β-catenin levels in abnormal pancreatic lesions in Smad4 mutant mice (57). The previous studies of β-catenin–mediated Wnt signaling are intriguing and suggestive of an important role of canonical Wnt signaling in pancreatic oncogenesis. However, Wnt signaling is slightly more complex than previously described, with various other non-canonical (i.e., not β-catenin–mediated) pathways capable of activation by Wnt ligand binding. One alternative intracellular signaling pathway, termed the non-canonical pathway, is mediated by intracellular calcium signaling and has been associated with various other co-receptors and intracellular mediators. Confusing the field of Wnt research is the fact that Wnt proteins are promiscuous in their ligand/receptor interactions, leading to a lack of specificity in ligand/receptor pairings. This makes it difficult to correlate a given Wnt or Frizzled with activation of a given downstream signaling cascade, as the role of co-receptors and intracellular mediators is crucial in guiding the activation of the canonical versus non-canonical Wnt pathway. However, there is a subset of noncanonical Wnts (e.g., Wnt 5a, Wnt 11) more prone to activation of the non-canonical pathways, mediated by calcium signaling and/or the planar cell polarity pathways, including MAPK signaling, rather than by the traditional β-catenin–mediated cascade (193–195). Interestingly, these non-canonical Wnts have also been implicated in pancreatic development and carcinogenesis. During development of the pancreas, Wnt 5a is believed to direct islet formation and positioning (196). More recently Wnt 5a overexpression as well as Wnt 5a inhibition has been examined in the context of pancreatic tumorigenesis. In these studies, Wnt 5a expression enhances cellular migration, proliferation, and invasiveness in several in vitro

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assays. In order to correlate these in vitro findings with in vivo changes, expression of Wnt 5a was examined in tumor specimens and noted to be elevated in PanINs and invasive pancreatic adenocarcinomas, suggesting a role for non-canonical Wnt signaling in these events (69). In these studies of Wnt 5a, CUTL1, a homeodomain family member and transcription factor, was also examined and found to act upstream of Wnt 5a (197). Interestingly, CUTL1 is known to play important roles in gene expression, morphogenesis, cellular differentiation, and cell cycle progression. CUTL1 is also known to interact with TGF-β and mediate some of the proinvasive effects of TGF-β later in tumorigenesis (198, 199). These findings then may be suggestive of a site of potential for cross-talk between TGF-β and Wnts, a convergence point of two distinct and very powerful signaling pathways, consistent with work in various other systems suggesting interaction and modulation of intracellular signaling by these two divergent pathways.

1.13.2

Notch

Notch proteins (Fig. 11.9) are a family of type I transmembrane proteins that function as receptors for membrane-bound ligands. All Notch proteins demonstrate many structural similarities, including an extracellular domain with multiple EGF repeats, as well as an intracellular domain with divergent domain types. Notch proteins are known to play a role in a variety of developmental processes and to control cell fate decisions by mediating interactions of physically adjacent cells. There are four known Notch genes in humans and two families of Notch ligands, Delta and Jagged (200). During development, Notch prevents cellular differentiation and maintains a population of undifferentiated precursor cells (201). A similar role has been proposed for Notch signaling in the setting of oncogenesis and maintenance of somatic stem cell pools in the adult. As is the case for Wnt signaling, the role of Notch signaling in cancer formation and progression has been characterized in great detail in various organ systems, for which the model of colon cancer is most relevant to our purposes. In this system the most notable feature of Notch pathway activation is lateral inhibition (187). Via lateral inhibition, a single cell is programmed to differentiate while its neighbors remain undifferentiated. This process of lateral inhibition mediated by Notch is also known to play an important role in pancreas development during embryogenesis (202), although Notch is also implicated in signaling via other distinct mechanisms during development of the pancreas as well (203). Using the intestine as a model system with well-characterized and defined roles of the various signaling cascades, the Notch, Delta, and Hes proteins in the intestine are primarily expressed in stem cell niches. As has been described, Wnt signaling is well established as a stem cell factor maintaining a pool of progenitor cells in the undifferentiated and highly proliferative state. In the intestinal crypts, the proliferating cells rely on a sequential combination of Wnt and Notch signaling pathways to maintain their proliferative activity. Interestingly, neither pathway in isolation is sufficient to maintain this proliferation. Temporally, Wnt signaling activates Notch signaling, whereas Notch

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Fig. 11.9 Notch signaling. Notch is a membrane spanning protein in which the extracellular domain interacts with a ligand bound to a neighboring cell. After ligand-ligand interaction, an ADAM family metalloproteases, tumor necrosis factor-α converting enzyme (TACE), cleaves the Notch protein, releasing the extracellular portion of the protein and allowing the cytoplasmic portion to be endocytosed. The intracellular portion of the Notch protein is then modified and eventually translocates to the nucleus, where it activates gene transcription in a complex with various transcriptional co-repressors/co-activators, such as CSL, to activate the expression of Notchrelated genes, such as Hes-1

seems to be permissive of the proliferative effect of Wnts. Wnt activation appears to drive the expression of Notch pathway components, whereas Notch activation mediates lateral inhibition within the population with active Wnt signaling (191). In models studying embryologic pancreatic development, suppression of Notch signaling results in a depletion of exocrine precursors and selective differentiation of endocrine cells. This observation is interestingly similar to the role of Notch in intestinal maintenance and oncogenesis, in which Notch mediates lateral inhibition as well as controls the secretory (goblet and enteroendocrine) phenotype. In the intestine, Notch is expressed by cells of the secretory lineage and inhibits neighboring cell differentiation into a similar phenotype. Notch signaling during development has also been demonstrated to play a role in maintenance of a progenitor population of cells. Many theories have surfaced regarding the role of Notch signaling and Notch reactivation in the adult pancreas. In general, evidence of Notch activation is absent in most normal adult pancreatic tissue. Typically, expression of the Notch target gene Hes-1 is limited to centroacinar cells and a small subset of ductal cells. However, during dysplastic transformation, increasing amounts of Hes-1 are observed in PanINs and pancreatic adenocarcinomas. This has led to the speculation that Notch activation may play a role in reactivation of progenitor cells,

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dedifferentiation of terminally differentiated cells, or some as yet undefined role in cancer progression and development (51). As activation of Notch signaling has been implicated in pancreatic adenocarcinoma formation, there has been much interest in the potential role of Notch signaling in controlling the behavior of pancreatic progenitor cells, maintaining pancreatic stem cell niches, or as an identifier of a progenitor population of cells. Examination of the Notch pathway has also focused on the influence of this signaling pathway in the process of oncogenic transformation. Various components of the Notch pathways are observed to be up-regulated in invasive pancreatic neoplasms. However, many of these observations of up-regulation of Notch and Notch-related genes in bulk tumor material or cancer specimens are suggestive of pathway activation, but are actually quite non-specific findings. Hes-1 is a basic helix-loop-helix transcription factor and is a known downstream mediator of Notch signaling. In pancreatic adenocarcinoma specimens, nuclear Hes-1 expression, indicative of Notch activation, is highest in basal epithelial cells of metaplastic ductal lesions, PanINs, and infiltrating ductal adenocarcinomas. Studies using immunohistochemistry also demonstrate up-regulated levels of Notch receptors and ligands in resected pancreatic adenocarcinoma specimens as well as in PanINs, and although suggestive of pathway activation these studies too lack functional implications. More functionally relevant studies in vitro suggest that EGF-mediated Notch overexpression in pancreatic explants induces dedifferentiation (58). In pancreatic adenocarcinoma, elevated levels of Notch proteins, as well as increased expression of the Notch mediator Hes-1, have been observed in various premalignant and malignant lesions (66). In human tissue samples increased expression of Notch mRNA and protein has been correlated with pancreatic cancer as compared with normal pancreatic tissue, and overexpression of Notch pathway components also results in increased levels of VEGF, suggesting cross-talk of this signaling pathway with other oncogenic signaling cascades (201). The Notch signaling cascade has been linked to many other important signaling pathways in pancreatic cancer formation. Recently, concurrent inhibition of the EGF and Notch signaling pathways has been demonstrated to result in decreased tumor cell proliferation and increased cellular apoptosis (204–206). Some of these effects of Notch inhibition occurred with resultant decrease in the activity of NF-kB and up-regulation of p21 and p27 (204). The combined implications of these various studies all suggest a role for Notch signaling in pancreatic oncogenesis, perhaps similar to the role played by Notch in intestinal carcinogenesis.

1.13.3

Hedgehog

Hedgehogs (Fig. 11.10) are a family of secreted proteins known to play key inductive roles during patterning of the ventral neural tube, the anterior-posterior limb axis, ventral somites, and foregut patterning during embryogenesis. The protein is made as a precursor that is autocatalytically cleaved and attached to the cell surface, thereby preventing it from freely diffusing. Defects in this signaling cascade have

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Fig. 11.10 Hedgehog signaling. Hedgehog proteins are capable of autocrine and paracrine signaling. The cell receiving the Hedgehog signals expresses the membrane-bound proteins Patched and Smoothened. In the absence of Hedgehog signaling, the Patched receptor suppresses the activity of a transmembrane receptor, Smoothened. Ptch interferes with the translocation of Smo to the membrane. In the presence of Hedgehog signaling and binding to the Patched protein, the Smoothened receptor is de-repressed, translocates to the cell membrane, leading to activation of this protein with resultant translocation of Gli protein to the nucleus and activation of gene transcription

been associated with holoprosencephaly (HPE), a disorder in which the developing brain does not appropriately separate into right and left hemispheres. It is also implicated in the VACTERL syndrome, characterized by vertebral defects, anal atresia, tracheoesophageal fistula, esophageal atresia, radial and renal dysplasia, cardiac anomalies, and limb abnormalities. These global effects on development speak to the importance of these signaling proteins as morphogens and patterning templates during embryonic organ formation. In addition to its role during development, the Hedgehog family of proteins has also been implicated in various models of oncogenesis. In pancreatic adenocarcinoma, Sonic Hedgehog (Shh) has been implicated in the initiation and maintenance of the process of carcinogenesis. Shh plays a key role in normal pancreatic development. It appears that downregulation of Shh is necessary for the induction of pancreatic-specific genes such as PDX1 (see the following). It also has been known to play a key regulatory role in rapidly proliferating epithelia. The role of Shh in the adult is perhaps best understood in the gastrointestinal system. In this system, Shh demarcates villi from crypts and is concentrated in the intervillous regions of the epithelium. The receptors for Shh (Patched 1 and 2) and the effectors of Shh signaling (Gli 1, 2, and 3) are restricted to the underlying mesenchyme. Conversely, Shh and Ihh expression

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are restricted to the epithelium. In the intestine, Shh and Ihh are produced only in sites where proliferation persists. Therefore, this occurs in intervillous pockets at the base of the crypts where the intestinal stem cell compartment exists. There is the suggestion that the Shh proteins diffuse outward from these sites and exert their effects at a distance. The theory proposes that they function to maintain proper spacing between the crypts, although they likely play a broader role in the proliferative and regenerative capacity of these cellular compartments as well (187). In pancreatic cancer Shh is misexpressed in a majority of PDACs and is identified early in the PanIN progression. Shh is thought to be activated in the context of inflammation and/or repair in response to toxic or caustic stimuli, since it is not normally expressed in the pancreas. Its in role early on may be in regeneration and protection by promoting a GI metaplasia of the pancreatic epithelium. In pancreatic cancer, however, Shh is believe to be important in tumor initiation. Misexpression of Shh in the pancreatic endoderm results in tubular formations that resemble human PanINs; however, activation of Shh signaling alone is not sufficient to induce cancer in a mouse model (59, 60). Shh is believed to play an equally important later role in PDAC, where it appears to be important for proliferation and survival at least in a subset of pancreatic cancer cell lines (59). It appears that Shh must cooperate with other mutations, such as K-ras, in order to promote and maintain pancreatic tumorigenesis (68). The mechanism by which Shh contributes to pancreatic tumorigenesis is now better understood. Shh expression results in enhanced proliferation of pancreatic ductal epithelial cells (PDECs) through MAPK and PI3K signaling pathways. Shh also has a protective effect of PDECs from apoptosis through a PI3K-dependent mechanism. In PDECs transfected with various combinations of expression constructs, cells expressing both K-ras and Shh undergo significantly increased levels of proliferation. Unexpectedly, this proliferative capability is not inhibited in the presence of inhibitors of the PI3K pathway. In addition, these cells are also refractory to Shh inhibition once the combined activation of K-ras and Shh has begun. Again, this suggests that K-ras and Shh may cooperate in initiation of tumorigenesis, but that these neoplasias may subsequently become pathway independent—and perhaps then chemoresistant. Reassuringly, simultaneous blockade of both cascades in these studies does suppress the oncogenic activity of these cells, perhaps revealing some level of cooperation and redundancy between the two signaling networks (59, 60, 68). Shh thus may contribute to pancreatic carcinogenesis by promoting proliferation, inhibition of apoptosis and enhancing chemoresistance.

1.13.4

PDX1

PDX1, also called insulin promoter factor 1 (IPF-1), is an islet-related protein known to activate transcription of the insulin and somatostatin genes. It is an important regulator of islet hormone expression (207, 208), as well as a key modulator of pancreatic development (209). There is an association between mutations in this gene and various disorders of the pancreas, including diabetes mellitus (DM) (210).

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During embryologic development, PDX1 is required for the initiating steps of pancreas formation, and the expression of PDX1 correlates with a population of cells capable of giving rise to all of the pancreatic cellular lineages (211). In the adult pancreas, PDX1 expression is normally limited to islet cells (208). However, PDX1 expression has also been observed in metaplastic ducts in TGF-α–induced models of pancreatic metaplasia as well as PTEN gene knock-out mice (51). Interestingly, PDX1 has also been observed to be weakly positive in PanIN lesions in K-ras mutant mice (212). Finally, PDX1 is also expressed in many human pancreatic cancers (1), and this expression correlates with poor patient outcome (213). Therefore, the expression of PDX1 in various malignant and premalignant lesions suggests either activation of an immature cell type, marked by PDX1 expression, or expansion of a cell type with re-acquired PDX1 expression, either of which may be important in pancreatic carcinogenesis (51).

2 Maintenance and Progression Factors 2.1

Cell Cycle Regulators

2.1.1

p16 (INK-4A) and p19 (ARF)

The tumor suppressor gene p16 is also known by several different names—INK4A, CDKN2, and MTS1 (Fig. 11.11). It has been observed that approximately 95% of pancreatic cancers have a loss of function of the p16 gene (48, 214, 215). The mechanism for this inactivation can occur by mutation, deletion, or promoter hypermethylation (91, 215–220). Conversely, mutations in p16 are very rare in the setting of chronic pancreatitis and, therefore, the absence of p16 in a subset of patients with pancreatitis may indicate a subpopulation at high risk for development of pancreatic adenocarcinoma (221). It is thought that mutations in p16 may play a role in the aggressiveness of pancreatic adenocarcinoma. It has been noted that tumors are larger and survival shorter in patients with a p16 mutation (218, 220). Mutations in p16 have also been found in the settings of gliomas, melanomas, leukemias, and esophageal squamous carcinomas (222). The locus that encodes the p16 protein is 9p21; however, this locus encodes two distinct proteins, INK4A/p16 and ARF/p19 in mice and ARF/p14 in humans, which can be classified into two separate cell cycle regulators (see Fig. 11.11A). Although INK4A and ARF are generated from the same locus, they do not share any amino acid homology. This is a result of a unique first exon (exon 1-β—ARF; exon 1-α—INK4a) being spliced onto common exons 2 and 3. Furthermore, unique translational reading frames result in proteins that share no homology. INK4Ap16 regulates G1-S cell cycle transition through regulation of RB inhibitors of cyclin-dependent kinases (e.g., CDK4 kinase). INK4Ap16 proteins prevent the phosphorylation of RB, preventing the release of E2F from RB and resulting in

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Fig. 11.11 A. INK/ARF locus. The INK4a/ARF locus encode for two distinct proteins. Each protein has a unique first exon INK4A, uses exon 1-α and ARF uses exon 1-β these are spliced on to common downstream exons. Different internal translational reading frames encode unique proteins with limited homology. B. Cell cycle. The cell cycle is composed of distinct phases (in yellow) including: (1) the G phase, divided into G0 (quiescent/senescent phase), G1a, G1b, G1c, and G1d; (2) the S phase; (3) the G2 phase; and (4) the M phase. The G0 phase is the quiescent or senescent phase of the cell cycle and is often found in terminally differentiated cells. The G1 phase extends from the previous mitosis and cytokinesis until the beginning of DNA synthesis. The S phase commences with DNA synthesis and ends when the amount of DNA has doubled, resulting in sister chromatids. The G2 phase occurs after the S phase until the next mitosis begins, and is a time dedicated to protein synthesis and microtubule formation. Finally, the M phase occurs; this encompasses the processes of mitosis and cytokinesis (cytoplasmic division). Various cyclin-dependent kinases and cyclins participate in this process, and there is a strict regulation system of various proteins that are responsible for halting cell cycle progression at sequential cell cycle checkpoints

cell cycle arrest in G1. In the absence of INK4Ap16, cyclinD1 in association with cyclin-dependent kinase 4/6 (CDK4/6) phosphorylates RB. Hyperphosphorylated RB (pRB) releases E2F, which then transcribes genes necessary for entry into

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S phase (see Fig. 11.11). INK4a appears to be dispensable for normal development and homeostasis, as loss-of-function mutations are not lethal (2, 223–225). Interestingly, germline mutations in p16 are associated with the familial atypical mole-malignant melanoma (FAMMM). Bearers of this mutation have a 12-fold increased risk of pancreatic cancer. However, pancreatic cancer is seen in some but not all FAMMM kindreds, suggesting that other mutations or modulating factors contribute to pancreatic cancer formation (216, 226). The alternate protein ARF has been identified to cause G1 arrest by several p53dependent mechanisms. First, ARF acts as a stabilization factor for p53 by sequestering MDM1, a protein active in p53 degradation. Second, ARF proteins interact with the E2F-1, -2, and -3 transcription activators to inhibit their transcriptional activity and induce their degradation via the 26S proteasome pathway in a p53dependent manner. The impact of ARF on the E2F proteins provides a mechanism for p53-independent ARF activity on cell cycle progression and tumor susceptibility (2, 89, 227). However, the finding that p53 (see below) is frequently mutated in tumors noted to have homozygous deletions of the INK4A locus suggests that ARF may have other p53-independent roles in pancreatic tumorigenesis. Overall, the current theory regarding the role of p16/p19 in pancreatic adenocarcinoma purports that these mutations are likely important in tumor maintenance and progression rather than tumor initiation, since there is such a low incidence of tumor formation in the setting of germline mutations and the sporadic mutations and subsequent malignancies seem to occur after the initiation of oncogenesis and after the activation of K-ras (221). Therefore, these mutations are often identified in PanIN2 or PanIN3 lesions as well as pancreatic adenocarcinomas, rather than in the earlier precursor lesions (217). It has been observed that tumors with K-ras activation are often the same subset of tumors in which associated mutations are also found in the p16/p19 genes. In the previously discussed mouse model of K-ras constitutive activation (91), these mutations were associated with PanINs but rarely with malignant lesions, whereas mutations in INK4A/ARF were not associated with malignancies. However, in the context of mutations in both K-ras and INK4A/ ARF, there was a very rapid evolution of high grade pancreatic neoplasias and subsequent death of the animals (91). This again supports the theory of K-ras as an initiator of neoplasia and INK4A/ARF as a maintenance or progression factor as well as a promoter of a less differentiated and more malignant phenotype (227a, 215, 224, 225, 228).

2.1.2

p21/WAF1

The potent cyclin-dependent kinase inhibitor p21 has also been called WAF1 and CIP1. p21 acts as a regulator of cell cycle progression at G1 by binding to and inhibiting the activity of cyclin-CDK2 or –CDK4 complexes (229, 230). The expression of this protein is tightly controlled by the activity of the tumor suppressor p53, as p21 mediates the p53-dependent cell cycle G1 phase arrest in response to stressful stimuli (231, 232). p21 Itself interacts with proliferating cell nuclear antigen (PCNA)

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to play a regulatory role in S phase DNA replication and repair (234–235). p21 Is also activated by caspases and plays a role in apoptosis following caspase activation (236, 237). Since its activity is associated with induction of apoptosis, it follows that its inactivation is associated with increased proliferation and decreased apoptosis, which is in keeping with observations in various experimental systems (232). In pancreatic cancer, inactivation of the p21 signaling pathway is often noted to coordinate temporally with the onset of K-ras mutations (233, 234). The MAPK pathway has been implicated in this p21 activation (2). There has also been observed up-regulation of p21 in the context of Notch signaling as well as Shh signaling (68, 112).

2.1.3

p53

Mutations in the tumor suppressor p53 are the most common somatic alteration found consistently in various human cancers. Hence, it is a very potent and yet very nonspecific proliferative and/or survival factor. P53 is a cell cycle regulator that is capable or causing G1 or G2 cell cycle arrest. It is well established that p53 plays a role in modulating the cellular response to cytotoxic stress, DNA damage, and/or hypoxia via either cell cycle arrest or apoptosis through an ARF-independent mechanism (235). In the pancreatic cancer progression model, p53 mutations occur later at the PanIN-3 stage. In PanIN3 lesions of the pancreas, loss of p53 has been associated with abnormal mitoses and severe nuclear abnormalities (30). In pancreatic cancer, p53 mutation can be found in 50–75% of tumors (236). These mutations appear to correlate with later lesions demonstrating more severe levels of dysplasia and invasiveness (2, 89). These observations imply that genomic instability may be an integral component of tumor progression at these more advanced stages, and this may be mediated in part by inactivation of p53 (see the following) (237). Many studies have demonstrated a synchronous interaction between progressive telomere dysfunction and p53 loss or inactivation in the progression of carcinogenesis (238). In various systems examining pancreatic adenocarcinoma, loss of p53 function is associated with inappropriate cell growth, increased cellular survival, and genetic instability (7, 237). Therefore, p53 is considered a maintenance or progression factor in pancreatic adenocarcinoma, rather than an inductive agent. In addition, this associated chromosomal instability and subsequent gene amplifications and/or deletions are manifestations of the heterogeneity of these tumors.

2.2

Genetic Instability: Progression Factor

2.2.1

Telomere Dysfunction

Telomeres are GC-rich DNA “caps” found on the terminal ends of chromosomes. They consist of highly repetitive DNA that allows transcription of DNA without truncating the end of the message. Telomeres allow faithful replication of DNA and

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protect against abnormal recombination events and against genomic instability. In general telomeres are sequentially shortened during each replication, and thus in normal somatic cells this phenomenon has been thought to be important for senescence. Telomeres can be extended by telomerases, such as telomerase reverse transcriptase (TERT), in order to maintain telomere length (239). In pancreatic cancer telomere dysfunction is thought to contribute to pancreatic cancer progression and maintenance by two mechanisms: (1) telomere attrition, and (2) telomerase activation. Shortened telomeres have been found in PanIN1 lesions and are thought to be early markers of neoplasia and important in tumor induction (26, 240, 241). It is thought that this telomere shortening and dysfunction may contribute to the formation of free ends that are very susceptible to chromosomal rearrangements and subsequent mutations through breakage-fusion cycles (242, 243). Abnormal breakage with chromosomal fusion results in abnormal chromosomes with two centromeres; thus during mitosis they are abnormally segregated. Histologically these are identified as anaphase bridges. The resultant daughter cell not only has abnormal genomic copy numbers, but also has free sticky ends that are highly recombinogenic. This loss of telomeres is of interest in systems in which the cells induced to proliferate are not normally proliferative cells (i.e., in settings of toxin damage or chronic inflammation), since these cells may not contain telomerase, the enzyme necessary for telomere extension and protection. Thus, early in pancreatic carcinogenesis telomere shortening is believed to contribute to pancreatic cancer progression by this genetic instability. Abnormal activation of telomerase in cancer cells enables them to maintain functional telomeres at the end of chromosomes, preventing them from senescence. Telomerase is consequently a key enzyme in the process of immortalization of cancer cells and is believed to play a pivotal role in carcinogenesis (244, 245). In general, telomerase activity is highly overexpressed in malignant cells, compared to normal cells (246). With regard to pancreatic cancer, in contrast to the very early alterations seen in telomere size and stability, telomerase activation is a late-stage alteration but one which is present in up to 95% of pancreatic adenocarcinomas (247). The mechanism for the misexpression of telomerase is poorly understood. Telomerase is a uniquely appealing target for drug discovery because its expression in normal versus cancer cells suggests it may be a selective target for cancer cells. A variety of approaches to telomerase inhibition are being investigated in human studies.

2.2.2

BRCA2

BRCA2 is a tumor suppressor protein with a well-established correlation with various cancer syndromes, but an incompletely understood mechanism of action (248). It is clear that BRCA1 and BRCA2 can activate transcription as well as participate in double-stranded DNA break repair and/or homologous recombination (248–250). Thus, mutation in this gene contributes to genetic instability in pancreatic cancer tumorigenesis. Inherited mutations in BRCA2

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have been associated with various breast and ovarian cancer syndromes (49, 248, 250); however, the penetrance for pancreatic cancer appears to be variable. Carriers with this mutation have a 10-fold higher risk for pancreatic cancer as compared to those without the mutation. BRCA mutations are found in 5% of pancreatic cancer patients with no family history and in 17% of those with a family history of pancreatic cancer (251). In terms of inherited mutations predisposing to pancreatic adenocarcinoma, BRCA2 is one of the most common mutations found in familial pancreatic cancer syndromes (252). In somatic cell acquisition of BRCA2 mutations, it appears to be a later event and to be restricted to the more severely dysplastic lesions, such as PanIN-3 lesions (253). Therefore, BRCA2 mutations are thought of as a progression factor likely due to enhanced genetic instability. BRCA-mediated genetic instability may require the inactivation of other tumor suppressor proteins concurrently, such as p53. Experiments in mice have shown that homozygous deletion of BRCA1 can be rescued by mutating p53. Furthermore, in a breast cancer model using serial BRCA, p53 gene mutation suggested that loss of p53 is critical for the development of BRCA associated tumors (254).

2.2.3

MicroRNA

MicroRNA are small non-coding RNAs in the size range of approximately 19–25 nucleotides in length. They are spliced into these small segments from larger 70to 100-nucleotide hairpin RNA molecules by an Rnase called Dicer. These small single-stranded RNA molecules have gained much interest in the last few years, even resulting in the Nobel Prize award to Andrew Fire and Craig Mello for their work in developing this field of research. These small microRNAs bind to their complementary RNA target, resulting in the degradation and suppression of this target mRNA’s expression. The interest in these molecules is fueled not merely by their activity in vivo (which is still being clarified), but also by their utility in vitro for the creation of siRNA, small molecules capable of silencing the expression of various RNA products. In the field of cancer research, these molecules have gained much popularity as proto-oncogenes by inhibiting the expression of various tumor suppressors. Several groups have recently utilized these findings to evaluate the microRNA signature of various tissues under different circumstances in order to find a “fingerprint” of the tumor state for identification, risk stratification, and treatment planning, as well as to gain a more basic understanding of the process of tumorigenesis. An interesting study by Bloomston et al. has recently demonstrated distinct patterns of expression associated with pancreatic adenocarcinoma, chronic pancreatitis, and normal pancreatic tissue with identification of miRNA markers associated with short- versus long-term survival (255, 256). These findings are intriguing in terms of potential diagnostic and risk-stratification tools. In addition, modulation of these miRNAs and identification of their targets may give much insight into the complex process of pancreatic oncogenesis.

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3 Conclusion Pancreatic adenocarcinoma is a disease that carries a significant societal burden in terms of incidence and mortality, and one for which our current therapies are still inadequate. Over the last several year new insights into theories of basic cancer formation such as the role of morphogenic gradients, tissue microarchitecture, and 3D structure on the subsequent activity of cancer cells in their tissue milieu are now better understood. The field of cancer research has expanded from its earlier focus solely on the cancer cell to a broader perspective, which includes the local tumor environment or tumor cell niche in models of cancer formation. It has become more and more clear that the local environment plays a modulatory role in tumor cell behavior, eventual tissue invasion, and metastasis. The signaling pathways involved in pancreatic cancer initiation and progression are also now better understood. Signaling proteins, such as K-ras, PI3K, and INK4a/ARF, were historically the focus of signaling research. Recently there has been an emphasis on new pathways, in particular on various developmental pathways, which are currently experiencing a resurgence of interest due to their role in oncogenesis and stem cell maintenance in various organ systems. The Wnt, Notch, and Hedgehog signaling pathways are only a few examples of these embryologic pathways. There are many intriguing findings suggesting key roles for these and other growth factor signaling pathways in directing local cellular behavior and oncogenic transformation. A further understanding of these cascades may facilitate the eventual design of more focused therapeutics for this malignancy. The end goal of all these studies is the eventual translation of this information into treatment of this dire disease, which carries such a poor prognosis. There are many trials currently underway targeting various signaling pathways, and we anticipate many new and novel approaches to the care and treatment of patients with pancreatic adenocarcinoma.

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Chapter 12

Pancreatic Cancer Genomics, Epigenomics, and Proteomics Michael Goggins

1 Introduction Extensive genetic and epigenetic alterations accumulate during the development and evolution of pancreatic cancer. In turn these alterations give rise to additional proteomics changes within the neoplasm, extensive changes in adjacent tumor stroma, and secondary effects in other biological compartments such as the circulation. This chapter describes the spectrum of genetic, epigenetic, RNA, and proteomic alterations observed in pancreatic ductal adenocarcinomas as well as their biological and clinical utility.

2 Pancreatic Cancer Genomics Pancreatic cancer is among the most genetically well-characterized cancers (see Chapter 1). Among the many genetic alterations that arise during pancreatic tumorigenesis are activation of oncogenes by mutation (K-ras,BRAF) and amplification (c-myc, AKT(1), cyclin E) and inactivation of suppressor genes (p16, p53, SMAD4, BRCA2, STK11, MKK4, and several of the Fanconi anemia genes) (1–5) (for further details see Chapter x). The list of genes that are mutated in pancreatic cancer is likely to increase once the pancreatic cancer genome sequencing project is completed. This project is similar in scope and importance as the recently completed colon and breast cancer genome project (6). The accumulation of these genetic changes appears to be accelerated by genetic instability that arises from two broad mechanisms. The most common form is chromosomal instability (CIN) that facilitates loss of whole chromosomes and contributes to aneuploidy, a hallmark of malignant growth (7). The molecular causes of CIN are not as completely understood but one important and common mechanism is accelerated telomere shortening that is evident in most pancreatic cancer precursors (PanIN). Shortened telomeres are thought to lead to sticky chromosomes and aberrant fusion breakage fusion cycles during cell division. Chromosome breakages that manifest as loss of heterozygosity (LOH) events and give rise to partial loss of the distal ends of chromosomes A.M. Lowy et al. (eds.) Pancreatic Cancer. doi: 10.1007/978-0-387-69252-4, © Springer Science + Business Media, LLC 2008

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also lead to sticky chromosomes. Certain genetic mutations such as mutations in the DNA repair genes BRCA2 and Fanconi anemia pathway genes and probably as yet unidentified mutations in DNA repair genes also likely fuel the development of chromosomal instability (8). Environmental causes of DNA damage such as cigarette smoking are also predicted to influence the development of CIN. In addition, cellular folate deficiency has been postulated as a cause of DNA damage, because anti-folates cause DNA breakages and folate sensitive fragile sites, and there is evidence that genetic alterations in the folate pathway could facilitate chromosomal losses in pancreatic cancers (9). DNA damage culminates in widespread chromosomal alterations in pancreatic cancers. These chromosomal alterations have been characterized in genome-wide studies characterizing pancreatic cancer deletions using hundreds of microsatellite markers (10). Using a similar strategy patterns of LOH have been identified in familial pancreatic cancers (200). More recently, similar results have been noted using SNP microarrays that have also revealed the extensive chromosomal duplications present in these cancers (11). Array CGH studies have also been used to accurately define genetic deletions and amplifications that occur in these cancers (12–16). The second form of genetic instability, microsatellite instability (MIN) is found in only a small percentage (~3%) of pancreatic cancers (17), but unlike CIN, the mechanism of MIN, inactivation of mismatch repair genes, is well understood. Mitochondrial DNA mutations occur during the development of pancreatic and other cancers but their functional significance is uncertain (18). Taking advantage of our expanding knowledge of pancreatic cancer genomics in the clinical setting is only beginning to occur. Understanding the genetics of familial pancreatic cancer can be rapidly translatable into patient care. For example, patients with a family history of pancreatic cancer and a germline BRCA2 mutation can undergo genetic counseling and may benefit from screening for evidence of pancreatic neoplasia. It has recently been determined that individuals with multiple relatives with pancreatic cancer can benefit from pancreatic screening using a combination of endoscopic ultrasound and computed tomography (19). Approximately ten percent of appropriately selected patients have significant precursor lesions in their pancreas such as intraductal papillary mucinous neoplasms and high-grade PanINs treatable with surgical resection. As our knowledge of familial pancreatic cancer susceptibility genes grows, so too will our ability to target the most at-risk individuals for screening. At the same time, screening of the pancreas using imaging identifies many subtle nonspecific alterations of uncertain significance. Accurate diagnostic markers of pancreatic neoplasia could aid in the diagnosis of suspicious pancreatic lesions identified by screening (19, 20, 21). Genetic markers of pancreatic cancer have the potential to be useful for molecular diagnostic assays. Although most genetic alterations are not suitable as diagnostic markers, sensitive markers such as mutant K-ras or and specific alterations such as p53 mutation have been evaluated using a variety of assays in an attempt to improve the diagnosis of patients suspected of having the disease. Mutant K-ras can be detected in sources distant from the cancer such as pancreatic juice, duodenal fluid, stool and blood using sensitive mutation assays (22–24). Unfortunately, K-ras

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mutations are not specific for invasive pancreatic cancer; mutant K-ras is found in pancreatic tissue among asymptomatic smokers and patients with chronic pancreatitis (23, 25, 26). Circulating mutant K-ras can be detected in serum in only a minority of patients with resectable pancreatic cancer (27, 28). Although these studies have limited the enthusiasm for mutant K-ras as a diagnostic marker, recently a sensitive assay have been developed that can quantify levels of mutant K-ras in blood and pancreatic juice (29). Similarly, improvements in assay performance could permit the use of mutant p53 and p16 to be used as a marker of pancreatic cancer. In this regard, recently described assay approaches such as “heteroduplex analysis of limiting dilution PCRs” (30) and other approaches may make this feasible (31). Finally, it is widely believed that certain genetic patterns in pancreatic cancer will ultimately be used for clinical decision making because their ability to predict outcome, particularly responses to therapies. In this regard, it is known that pancreatic cancers with inactivation of the BRCA2/Fanconi pathway are sensitive to mitomycin C in vitro, analogous to the DNA repair defect in patients with Fanconi anemia (32), and the significance of this observation is being tested in a clinical trial.

3 Epigenetics Epigenetic alterations, including DNA hypermethylation and hypomethylation, and the associated transcriptional changes of the affected genes are central to the evolution and progression of various human cancers, including pancreatic cancer (33, 34). In human cells, methylation of DNA is restricted to the cytosines at CG dinucleotides. CG dinucleotides (often referred to as CpGs) are often found in regions of DNA rich in CG dinucleotides known as CpG islands. CpG islands are commonly located in the 5¢ regulatory regions of genes, and most CpG islands are normally unmethylated. With the use of genome-wide screening technologies as well as conventional candidate gene approaches, many genes have been identified that are affected by aberrant DNA methylation in pancreatic cancer. Importantly, the detection of DNA methylation alterations have been proposed for cancer risk assessment, the early detection of cancer, as well as for tumor classification, prognostication, and as therapeutic targets (35–40). Despite advances in our characterizing of cancer DNA methylation alterations, the etiology of these alterations is not well understood.

3.1

The Etiology of Aberrant DNA Methylation in Human Cancer

Multiple mechanisms are likely to be responsible for the full spectrum of methylation alterations associated with cancer. Overall, we and others hypothesize that alterations in methylation in cancers likely reflect the combined effects of aging,

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nutritional and environmental influences, imprinting effects, genetic alterations associated with tumor development and cell environmental changes due to tumor stromal interactions or chronic inflammation (41–51). Some of these changes could influence the regulation of normal DNA methylation and chromatin modifications, but many of these changes would reflect changes at the local gene level in the context of normal epigenetic machinery. It remains unknown which of these mechanisms dominate. One likely scenario is that DNA methylation initially arises in discrete CpG sites independent of gene expression but then spreads into promoter CpG islands presumably through a loss of balance between factors that promote and those that protect against methylation spreading. This model was supported by a study that GSTP1 methylation in prostate cancer cells is initiated by a combination of transcriptional gene silencing (by removal of the Sp1 sites) and seeds of methylation that subsequently spreads across the promoter CpG island (52). It has been also suggested that establishing transcriptional silencing of a gene involves a close interplay between DNA methylation and histone modifications, and that the DNA methylation change in cancer can be a secondary event that may occur as a consequence of genetic or other events, such as loss of transcription factor(s), that alter the transcriptional activities of affected promoters. For example, the leukemiapromoting PML-RAR fusion protein has been shown to induce the RARA gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters (53). Another example is revealed by the loss of estrogen receptor expression by RNA interference which results in silencing of downstream target genes through recruitment of polycomb repressors and histone deacetylases to their promoters, followed by a progressive accumulation of DNA methylation in their promoter CpG islands (44). It also appears that there are sequence variations in the human genome that create susceptibility differences by predisposing the site to epigenetic changes (54). Transposons are prone to methylation, as exemplified by the agouti mouse (45–48), chromosomal regions are targets for methylation in cancer (55). In certain model systems histone modifications (methylation of histone H3 lysine-9) are the primary events that occur prior to DNA methylation (49). These findings have suggested that, at least at some genetic loci, initial silencing events lead to chromatin modifications that may predispose promoter CpG islands to hypermethylation. In certain experimental settings, alterations in chromatin insulator function can give rise to methylation of imprinted or other genes (50).

3.2

Aberrant Hypermethylation in Pancreatic Cancer

Aberrant DNA hypermethylation in pancreatic cancer was first reported in 1997 by Schutte et al. who demonstrated aberrant hypermethylation and gene silencing of the p16/CDKN1A gene in a subset of pancreatic cancers that harbored wild-type p16 alleles (56), suggesting DNA methylation as an alternative pathway to inactivate this important tumor-suppressor gene in pancreatic cancer. Ueki et al. analyzed a large panel of 45 pancreatic cancers for methylation status of multiple genes (including

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p16 and hMLH1) and CpG islands previously identified as aberrantly methylated in other cancers (57). The results also revealed that a small subset of pancreatic cancers have a higher prevalence of DNA methylation, suggestive of the presence of CpG island methylator (CIMP) phenotype (58–60). Subsequent studies have demonstrated that pancreatic cancers with methylation of the highest proportion of CpG islands in a gene panel are larger in size and are found in older patients (59), although distinct biological, clinical, or pathologic differences have not yet been identified to support the use of a CIMP classification for pancreatic cancer. Nonetheless, numerous studies in recent years have demonstrated that methylationinduced silencing of biologically relevant genes in pancreatic cancer is common and influences tumor behavior. Indeed, many investigators have used candidate gene approaches to identify various tumor-suppressor or cancer-related genes that undergo aberrant methylation in pancreatic cancer, including TSLC1/IGSF4 (61), SOCS-1 (62), cyclin D2 (63), WWOX (64), DUSP6 (65), and HHIP (Hedgehog interacting protein) (66). Genome-wide screening techniques have aided the discovery of epigenetic alterations in pancreatic cancer. For example, methylated CpG island amplification coupled with representational difference analysis (MCA/RDA) was used to isolate a number of CpG islands differentially methylated in pancreatic cancer (59). One of the CpG islands identified was located in the 5Î region of the gene preproenkephalin (ppENK), encoding for a native opioid peptide with growthsuppressor properties, which was found to be aberrantly methylated in the vast majority (>90%) of pancreatic cancers (59). Aberrant methylation at p16, ppENK, and other genes was also detectable at various frequencies in precursors to invasive adenocarcinoma known as pancreatic intraepithelial neoplasia (PanINs) (67, 68) and in intraductal papillary mucinous neoplasms of the pancreas (IPMNs) (69), with increasing prevalence with advancing stage of the neoplasms (69, 70). Global gene expression profiles have been used to search for novel methylated genes in pancreatic cancer (71, 72). For example, a global gene expression comparison of IPMNs and normal pancreatic ductal epithelium identified CDKN1C/p57KIP2 as a gene commonly down-regulated in pancreatic ductal neoplasms through CDKN1C promoter hypermethylation and histone deacetylation, and/or loss of the CDKN1C expressing allele as evidenced by LOH of the CDKN1C locus and hypomethylation of LIT1, an imprinting control region that silences CDKN1C when LIT1 is hypomethylated and expressed (73). Another approach termed “differential and epigenetic gene expression profiling” has been used to prioritize candidate genes for methylation analysis (74). After genome wide analysis has generated a list of candidate methylated genes, prioritizing candidates to genes that are markedly underexpressed in primary pancreatic neoplasms can facilitate the identification of important genes (74). Other useful microarray-based approaches for discovering methylated regions include the use of “CpG island microarrays” (75) and promoter arrays (76–78), that have also been applied to detect genes specifically associated with methyl-CpG binding proteins (79) or those with histone modifications (80, 81). Functional consequences of aberrant hypermethylation in pancreatic neoplastic progression. Several epigenetically silenced genes identified in pancreatic cancer are thought to be functionally involved in tumor growth, invasion, metastasis, and

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chemoresistance (Table 12.1). For example, the classic tumor suppressor gene p16 undergoes methylation induced silencing in almost all those cancers (~15%) that do not have biallelic inactivation of p16. Many other aberrantly methylated genes possess anticancer functions, but are only silenced epigenetically in human cancers. For example, epigenetic silencing of the gene RELN occurs in cancer cells and influences pancreatic cancer cell migration and invasion properties (74). RELN was originally identified as a gene responsible for the mouse reeler phenotype a model of brain malformation (82). Loss of reelin function through mutations causes lissencephaly (or “smooth brain”) (19), and decreased reelin expression has been associated with major psychiatric disorders, including schizophrenia, depressions, Table 12.1 Select genes aberrantly hypermethylated in pancreatic cancer

Gene

Known or predicted Chromosome function

Prevalence of DNA methylation in pancreatic cancer cell lines

Prevalence of DNA methylation in primary or pancreatic cancers

RELN SPARC

7q21 5q31.3-q32

6/9 (67%) 16/17 (94%)

23/32 (72%) 21/24 (88%)

9/10 (90%)

8/10 (80%)

4/17 (24%)

25/91 (27%)

3/9 (33%)

5/36 (14%)

7/9 (78%)

Not determined

11/11 (100%)

43/47 (91%)

6/19 (32%)

13/60 (22%)

14/17 (82%)

102/140 (73%)

2/16 (13%) 20/22 (91%)

5/12 (42%) 16/20 (80%)

10/20 (50%)

33/70 (47%)

0/9 (0%)

2/36 (6%)

5/9 (56%) 19/22 (86%) 14/22 (64%)

4/36 (11%) 71/109 (65%) 15/20 (75%)

21/22 (95%)

20/20 (100%)

BNIP3 TSLC1/IGSF4

CDKN1A/p16 CDKN1C/ p57KIP2 ppENK SOCS-1

TFPI-2 DUSP6 Reprimo HHIP

MLH1 RARβ cyclin D2 FOXE1 NPTX2

Neuronal migration cell—matrix interaction, Cell growth inhibition 10q26.3 Hypoxia-induced cell death 11q23.2 Cell–cell, cell– matrix interaction 9p21 Cyclin-dependent kinase inhibitor 11p15.5 Cyclin-dependent kinase inhibitor 8q23-q24 Neuropeptide precursor 16p13.13 inhibitor of JAK/ STAT pathway serine 7q22 Proteinase inhibitor negative 12q21-q22 Regulator of MAPK 2q23.3 p53-induced G2/M Cell cycle arrest 4q28-q32 Negative regulator of hedgehog pathway 3p21.3 DNA mismatch repair 3p24 Cell growth control 12p13 cell cycle control 9q22 Thyroid transcription factor 7q21.3-q22.1 Neuronal transport

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and autism (19–21). Another methylated gene, SPARC, is silenced in most pancreatic cancers in association with aberrant methylation and has inhibitory effects on the growth of pancreatic and other cancers in vitro and in vivo (83). Interestingly, SPARC is over-expressed in stromal fibroblasts adjacent to cancer cells and its expression in these fibroblasts may be regulated through tumor–stromal interactions (84), and patients whose peritumoral stromal fibroblasts express SPARC have a poorer prognosis than those without such expression (85). WWOX (WW domain containing oxidoreductase), a candidate tumor suppressor gene that maps to the common fragile site FRA16D, was recently shown to be inactivated in pancreatic cancer by genetic (deletion) and/or epigenetic (promoter hypermethylation) mechanisms, and transfection of WWOX induced apoptosis and inhibited colony formation of pancreatic cancer cell lines lacking the expression (64). Another gene of interest is TFPI-2 (tissue factor pathway inhibitor 2), encoding for a broad-spectrum serine proteinase inhibitor, which was found to be commonly inactivated by aberrant methylation in pancreatic cancer (70). Restored expression of TFPI-2 in nonexpressing pancreatic cancer cells resulted in marked suppression in their proliferation, migration, and invasive potential in vitro (70). BNIP3, a hypoxia-inducible proapoptotic gene, is silenced in pancreatic cancer, and loss of BNIP3 function may increase cellular resistance to hypoxia-induced cell death and certain chemotherapeutic agents including gemcitabine (86, 87). These studies highlight the role of epigenetic inactivation in the biological phenotypes of pancreatic cancer. On the other hand, it is also notable that genes whose expression is thought to favor neoplastic progression, such as COX-2 and CXCR4, have also been shown to be silenced in association with promoter hypermethylation in a subset of pancreatic and other cancers (84, 88). The biological significance of aberrant methylation at these potential cancer-promoting genes is unknown, but this phenomenon probably reflects a genome-wide propensity of CpG island hypermethylation that occurs during pancreatic carcinogenesis and highlights the fact that the mere presence of gene methylation and epigenetic silencing in a cancer does not necessarily mean that such silencing is functionally important.

3.3

Gene Hypomethylation in Pancreatic Cancer

DNA hypomethylation is another major form of epigenetic alteration in human cancer (89). This epigenetic alteration is detected both at genomic level (global hypomethylation) and at specific sequences (regional or site-specific hypomethylation) such as normally methylated repeat sequences and 5¢ regions of certain genes. Global DNA hypomethylation has been linked to genetic instability (90) and tumorigenesis (91–94). Interestingly, the effect of global hypomethylation on tumorigenesis is tissue and stage dependent. In some tissues such as the intestine, hypomethylation associated with reduction in DNA methyltransferase activity increases initial tumor development but reduces later tumor development (91–93). As global DNA hypomethylation is known to occur as a result of genetic and nutritional

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alterations in vitamin B12 and folate metabolism, but available evidence indicates that these factors are not responsible for the global hypomethylation found in most cancers. However, deficiency of nutrients essential for methylation such as vitamin B12 and folate is associated with an increased risk of several cancers, including pancreatic cancer (95, 96). In addition, we have found that pancreatic cancers with the most deficient methylene tetrahydrofolate reductase (MTHFR) genotypes have more DNA hypomethylation and more chromosomal losses supporting the hypothesis that global hypomethylation can promote genomic instability (9). Gene-specific hypomethylation at specific CpG sites has been linked to overexpression of affected genes and has led to an attractive hypothesis that promoter hypomethylation and associated epigenetic alterations in chromatin can cause gene activation. Methylation analysis of genes with differing expression status in pancreatic cancer demonstrated frequent hypomethylation at genes that are overexpressed in the neoplastic epithelium of pancreatic cancers and not expressed in normal pancreatic ducts (71, 97). It is not certain at this time if gene-related hypomethylation is a cause or consequence of altered transcriptional activity in cancer cells (89).

3.4

Diagnostic Potential of Epigenetic Markers in Pancreatic Cancer

The development of early detection strategies using molecular markers should lead to an improved clinical outcome for pancreatic cancer (98). DNA methylation abnormalities hold promise as novel screening/diagnostic markers of pancreatic cancer (36), especially for high-risk individuals such as those with a strong family history of pancreatic cancer (99). Aberrant methylation changes at CpG islands are readily identifiable in clinical samples with the use methylation-specific PCR (MSP) (100). MSP has been used successfully to identify methylated DNA in most biologic fluids, including blood, fine-needle aspirates (FNAs), saliva, prostatic fluid, and sputum of patients with cancers (36). In addition, quantification of the methylated DNA using real-time MSP may help to accurately distinguish cancers from non-neoplastic diseases. The diagnostic potential of epigenetic markers has been evaluated in clinical samples (i.e., pancreatic juice) from patients with different pancreatic diseases (72, 101, 102). Quantifying pancreatic juice methylation using real-time quantitative MSP (QMSP) using a panel of genes known to be aberrantly methylated (e.g., SPARC, ppENK, FOXE1, NPTX2, and CLDN5) could better predict pancreatic cancer than detecting methylation using conventional MSP (102). Recently, the diagnostic accuracy of several of these markers has been confirmed by other investigators (103–106). In one study found patients with pancreatic cancer had higher levels of p16 promoter methylation in their pancreatic juice samples, than controls with benign biliary disease and pancreatitis (31). Although the potential of using aberrantly methylated DNA as cancer markers is promising, several biologic features of DNA methylation need to be considered before it can be applied to clinical practice. One potential obstacle of using DNA

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methylation as a diagnostic tool in the pancreas is the problem of tissue-specific differences in normal methylation patterns. For example, many genes that undergo methylation in pancreatic cancer are normally methylated in the non-neoplastic duodenum, albeit at low levels in most cases, and, therefore, such methylation is frequently detected in duodenal secretions of patients with and without pancreatic cancer (63, 107). In addition, for some genes, the likelihood of finding methylation increases with patient age (59, 63, 107). Another concern with PCR-based assays such as MSP is one that was observed with mutant K-ras gene detection assays, which is that they can be “too sensitive”—the detection of a few methylated copies of DNA may identify lesions of low or even no malignant potential. For example, low-grade PanINs develop with increasing frequency in the pancreata of people as they age (108), and some of these PanINs harbor methylation changes that are not found in normal pancreatic epithelium. Despite this theoretical concern, initial studies quantifying pancreatic juice methylated DNA reveal readily distinguishable differences in methylation between patients with and without pancreatic cancer (102). From a standpoint of risk assessment, the observation that patients with pancreatic cancer have a greater propensity to methylate non-neoplastic duodenum, specifically at certain CpG islands, than patients without neoplasia is potentially useful (107). Additional studies are needed to identify the best set of methylation markers for early detection and/or risk assessment, determine detection technologies best suited for each application as well as their cost performance in the clinical settings, establish the sensitivity and specificity of these selected markers in larger studies.

3.5 Epigenetic Alteration as a Therapeutic Target in Pancreatic Cancer Cancer associated epigenetic alterations are attractive therapeutic targets because such epigenetic alterations, unlike genetic changes, are potentially reversible. Inhibitors of DNA methylation and HDAC have been shown to suppress tumor growth in vitro and in vivo, and some of the inhibitors are being tested in clinical trials for patients with different types of solid and hematological cancers (40). One DNA methyltransferase inhibitor, Decitabine has been extensively investigated for its effects on gene expression and for its antineoplastic potential. It is FDA approved for the treatment of myelodysplastic syndrome (109). The drug though has minimal activity in most solid tumors, and only causes modest reductions in DNA methylation in patients (110). Newer DNA methyltransferase inhibitors are undergoing evaluation. Similarly, HDAC inhibitors are also undergoing extensive clinical trials for a variety of cancers (111). SAHA has been FDA approved for orphan drug status for the treatment of lymphoid neoplasms (112). Only a few studies, however, have addressed the effects of epigenetic modifying drugs in patients with pancreatic cancer (113). The efficacy of these epigenetic modifying drugs may vary among individual cancers, and could be determined by the balance between activation of tumorsuppressor genes and that of cancer-promoting genes. Furthermore, a recent study

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showing that global DNA hypomethylation can lead to tumor formation in mice raises a question about the rationale for the use of demethylating agents for cancer. Thus, these questions need to be further investigated before DNA methylation and HDAC inhibitors are moved into clinical use for patients with pancreatic cancer.

4 RNA Alterations in Pancreatic Cancer The expression of hundreds of genes is altered during pancreatic cancer development, and knowledge of such alterations has been used to identify upstream genetic and epigenetic alterations and downstream protein alterations in pancreatic cancer (73, 114, 115). Identifying the protein products of these overexpressed genes may identify suitable targets for protein-based diagnostic tests. In addition, the direct assaying of pancreatic specimens for changes in specific RNA levels has the potential to assist in diagnosis. Such an approach is important because many of the proteins overexpressed in pancreatic and other cancers are not secreted proteins and therefore are not likely to be found in the circulation. Messenger RNA levels in samples to be screened can be quantified with the use of quantitative reverse transcriptase PCR or global gene expression profiling using chip-based assays. The promise of discovery using gene expression profiling has led to many publications describing differentially expressed genes in primary neoplastic pancreatic tissues compared with normal pancreas (114, 116–122). However, many tissue gene expression studies are fundamentally flawed because investigators fail to accommodate in their experiments the fact that many of the RNA profiles of a pancreatic cancer arise from expression changes in non-neoplastic stromal cells and normal pancreatic tissue is largely composed of acinar cells and not ductal epithelial cells. Many of the apparent gene expression differences identified are simply the result of comparing two different cell types. For this reason, it is critical to determine the cell of origin of genes tentatively identified as overexpressed in pancreatic cancer. Assays that can evaluate cell morphology, such as immunohistochemistry or in situ hybridization are helpful in this regard, and have been used to identify expression alterations that are unique to the peritumoral compartments (123). Tissue microarrays have been used to efficiently characterize genetic and protein changes of pancreatic and other cancers in a high throughput manner (124–132). Direct microdissection of individual neoplastic and normal ductal epithelium using laser capture techniques can largely overcome the confounding effects of mixed cell populations in tumor masses. Since the limited amounts of RNA from microdissected tissues requires additional amplification prior to array analysis that can lead to amplification bias, validation of RNA expression differences using independent methods is important. Many of the gene expression differences identified in pancreatic cancer have been evaluated for their potential diagnostic use. Perhaps the best-studied RNAbased marker to date has been the human telomerase reverse transcriptase gene (hTERT). About 90% of cancers express the telomerase subunit hTERT, and ~90% of patients with pancreatic cancer have detectable telomerase activity in their

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pancreatic juice (133). Although the detection of telomerase enzymatic activity or the hTERT subunit may be helpful in differentiating pancreatic cancer from benign pancreatic diseases, telomerase is also expressed in inflammatory cells limiting its use in routine clinical practice. The use of gene expression profiling coupled with confirmatory analysis of expression changes in the tissues has led to the identification of hundreds of genes that are overexpressed in pancreatic cancer cells relative to normal pancreas. Examples of such genes include mesothelin, prostate stem cell antigen (PSCA), 14– 3-3 sigma, S100P, S100A4, osteopontin, MMP-7, Muc4, kallikrein 10, fascin, and keratin 17 (84, 117–119, 134–138). The protein products of some of these genes have been evaluated as serum markers of pancreatic cancer including MIC-1, osteopontin, gp-39, HIP, TIMP-1 (139–145). For example, mesothelin and PSCA are expressed in most pancreatic cancers, and immunolabeling for mesothelin and PSCA has been evaluated as an aid in the interpretation of diagnostically challenging pancreatic biopsies (146, 147). It is likely that the expression patterns of other overexpressed genes in cytologic specimens, identified with the use of such approaches as immunohistochemistry, quantitative reverse transcriptase PCR, and gene chip profiling, could be used to distinguish FNAs of pancreatic cancers from noncancerous aspirates. Overall, only a minority of the protein products of overexpressed genes are generally suitable for use in serologic diagnosis. In addition, although some genes may be abundantly expressed in pancreatic cancer cells relative to the normal pancreas, in other tissues, the expression of such genes may be normal. Finally, many genes that are overexpressed in pancreatic cancer cells are also overexpressed in chronic pancreatitis tissues (120). Therefore, a careful characterization of the genes whose overexpression is most sensitive and specific for cancer is needed when assays to detect the protein products of overexpressed genes are designed. Ideally, a serum-based marker of pancreatic cancer should be a secreted protein overexpressed in pancreatic cancers, not expressed in non-neoplastic pancreas, and having a restricted pattern of expression in other organs and tissues. Despite the extensive characterization of pancreatic cancer gene expression profiles, no ideal candidate overexpressed gene has emerged as a suitable marker. One gene that fulfills some of these criteria is macrophage inhibitory cytokine (MIC-1). MIC-1 is overexpressed in pancreatic cancer cells relative to normal pancreas, and the protein product of MIC-1 is secreted. We have found serum MIC-1 to be more sensitive than CA19–9 for patients with resectable pancreatic cancer, although it is not more specific than CA19–9 (140). These characteristics could make serum MIC1 helpful in the early detection of pancreatic cancer in high-risk populations such as those undergoing pancreatic screening with endoscopic ultrasound (EUS) and computed tomography (CT) (20, 148), who have a modest pretest probability of disease. Since these patients are undergoing regular pancreatic evaluation, the consequences of an initial false-positive test are less than for an average risk person. In principle, in the absence of other diseases that elevate MIC-1, elevated MIC-1 levels could be used to prioritize subjects to further evaluation using more specific pancreatic imaging tests. Of interest, the combination of serum CA19–9 and MIC-1 measurements better predicted patients with pancreatic cancer than the use of CA19–9 alone (140,

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143). Another potential marker identified through gene expression profiling is osteopontin. Osteopontin is expressed in peritumoral macrophages and is usually increased in the serum of patients with pancreatic cancer (142). Infiltrating pancreatic cancer cells also induce changes in adjacent peritumoral cells. These peritumoral cells such as fibroblasts and acinar cells can obscure the specific expression changes that arise in pancreatic cancer cells when bulk pancreatic tissues are profiled. However, investigators have also sought to identify molecular alterations in peritumoral cells that could be useful markers and also determine how peritumoral cells influence tumor progression. Gene expression studies of microdissected peritumoral acinar cells indicate that several acinar derived proteins (HIP/PAP and gp39) are elevated in the serum of patients with pancreatic cancer (144). Similarly acinar derived proteins have been identified using direct proteomics approaches (144, 145). Direct characterization of cancer associated fibroblasts is more difficult because they are not easily microdissected, but in vitro studies of primary cancer fibroblast cultures have provided insights into how fibroblasts may influence tumor progression (149). More recently pancreatic cancer investigators have moved from expression profiling pancreatic neoplasms to investigating the transcriptional changes associated with specific pathways or genetic alterations such as K-ras mutations (150).

4.1

Gene Expression Alterations in Pancreatic Cancer Stroma

Cancer-associated stroma is thought to have a net tumor-promoting effect (151, 152). Pat Brown and colleagues have demonstrated that the gene expression phenotype of cancer associated fibroblasts helps predict outcome of patients with certain cancers (153–155). Recent studies by Polyak et al. indicate a role for epigenetic alterations in breast cancer fibroblasts (149). SPARC is associated with outcome among patients undergoing surgical resection for pancreatic cancer (85). We previously identified SPARC (secreted protein acidic and rich in cysteine, or osteonectin/BM40) as a gene that is frequently epigenetically silenced in pancreatic cancers (83). SPARC is a calcium-binding protein that interacts with extracellular matrix and influences cell migration, proliferation, angiogenesis (especially during wound healing), matrix cell adhesion, and tissue remodeling (156). SPARC is regarded as a marker of activated fibroblasts (157). SPARC knockout mice grow cancers faster than mice expressing SPARC highlighting its growth inhibitory functions (158, 159). When evaluating SPARC expression in primary pancreatic cancers, we observed different stromal SPARC expression patterns.

4.2

microRNA Alterations in Pancreatic Cancer

MicroRNAs are small 22 nucleotide noncoding RNAs that function as posttranscriptional regulators. Impressively, ~20% of all genes are microRNA targets. MicroRNAs are formed from larger hairpin RNA transcripts that are

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degraded by an the RNA enzyme DICER, then associate with an RNA-induced silencing complex (RISC) and bind to the 3¢ untranslated regions of many genes thereby inducing RNA degradation or translational repression. Over 400 hundred microRNAs have been identified and widespread alterations in these microRNAs have been identified in various types of cancer including pancreatic cancer (160–164). MicroRNA alterations in cancers can be classified according to the developmental origin of the tissue (165). While the expression of most microRNAs appear to be reduced in cancer, several microRNAs are overexpressed. The mechanism of these microRNA alterations in human cancers is largely unknown, although epigenetic suppression of microRNA has been described (166).

5 Proteomics Alterations of Pancreatic Cancer Proteomics is the large-scale analysis of unknown proteins in biologic fluids or cells. Proteomics studies are studies that use direct characterization of proteins in contrast to global gene expression profiling, which can be used to prioritize protein expression studies. Direct proteomics strategies have certain advantages. First, there may be significant dissociation between overexpressed transcripts and overexpressed proteins; indeed such discordance has been reported in comparisons of global transcriptomic and proteomic analyses of other human cancers (17). Second, because most expression techniques use “bulk” homogenates of cell lines and/or tissues, there is no preferential enrichment for the secreted class of proteins (i.e., the cancer “secretome”) (18), the portion of the proteome expected to harbor the most promising biomarkers. Indeed, the analysis of bulk tissues that contain mixtures of different cell types can lead to incorrect assignment of the expression status of RNAs or proteins (167). Furthermore, although global gene expression profiling of human tissues has led to the identification of important protein alterations in pancreatic cancer, only a few such proteins show promise as biomarkers. A major goal for investigators attempting to identify pancreatic cancer diagnostic markers is to identify the equivalent of a “PSA test” for pancreatic cancer. Although gene expression profiling has been a useful tool for identifying proteins overexpressed in pancreatic cancers such as mesothelin, PSCA, MIC-1, and osteopontin, the direct analysis of unknown proteins in pancreatic cancer tissues is important because many novel cancer protein alterations are post-transcriptional alterations that would not be evident from RNA analysis. Direct and large-scale analysis of proteins using proteomics approaches has the potential to overcome some of the limitations of relying on gene expression analysis, although these approaches come with their own limitations. Proteomics strategies are considered particularly useful for characterizing tissue samples that can not be characterized using gene expression approaches such as secondary fluids like serum, plasma or pancreatic juice. RNA profiling approaches do not readily distinguish secreted from nonsecreted proteins.

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A variety of proteomics approaches have also been utilized in an attempt to identify protein markers of pancreatic cancer (167–183). Proteomics studies identify hundreds of proteins and protein fragments. With this in mind, the Human Protein Reference Database (HPRD) (http://www.hprd.org) was developed to serve as a comprehensive collection of protein features, post-translational modifications and protein–protein interactions. Several groups have identified protein fragments in serum using the mass spectrometry technique known as surface enhanced laser desorption ionization (SELDI) and some of these protein fragments appear to perform as well or better than serum CA19–9 as a diagnostic marker (141, 184). Several peptides we identified as diagnostically promising have also been identified by others (185, 186). There can be problems with assay reproducibility using SELDI unless appropriate technical and analytical measures are taken (186–189), in part reflecting the complexity of the proteome and their sensitivity to alterations (188, 189). Another mass spectrometry approach utilizes matrix associated laser desorption ionization (MALDI) and has been used to identify pancreatic cancer proteins in serum. One interesting approach utilizes stable isotope labeling with amino acids in cell culture (SILAC) to compare the proteins secreted pancreatic cancer cells from non-neoplastic pancreatic ductal cells (182). Antibody microarrays are a promising discovery strategy for profiling serum proteins and have the potential to profile hundreds of protein in sera simultaneously (190). Another approach has been to selectively characterize serum glycoproteins (170). Pancreatic juice has also been investigated as a source of pancreatic cancer markers. For example, investigators have used one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry as well as SELDI to identify potential markers of pancreatic cancer (191, 192). Several groups have applied proteomics approaches to pancreatic cancer cells rather than secondary sources such as serum and compared them with normal and inflamed pancreas (193, 194). This approach has the advantage of evaluating proteins from the source tissue, but since primary tissues reflect a mixture of multiple cell types, determining the relative abundance of proteins in one tissue versus another can be a challenge. On the other hand, many of the proteins identified from serum proteomics studies are high-abundance proteins, and many of the differentially expressed proteins identified are not suitable for diagnostic use. Thus, despite much effort by proteomics investigators to identify markers of pancreatic and other cancers by profiling proteins, only limited success has been achieved to date in identifying markers that will ultimately be useful as diagnostic tests. This limited success highlights the challenge of interrogating the large numbers of proteins in the proteome (hundreds of thousands of proteins and protein modifications), their complex biology, the vexing chemical properties of proteins that can make them hard to separate, and the influence of physiologic states, sample collection, and sample handling can have on protein profiles (195, 196). For example, proteolysis of serum and other samples can continue after collection, ex vivo due to the continued activity of circulating proteolytic enzymes (197). Another problem that may arise when undertaking marker discovery studies is bias, wherein markers are identified that purport to reflect differences between patients with disease versus those

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without, but merely reflect trivial differences in the study populations such as patient age, or differences that arise from differences in sample handling or data interpretation (198). The problem of bias has been prominent in proteomics studies, which uses a highly sensitive discovery technology that does not easily distinguish artefactual and nonspecific alterations from biologically relevant ones. Despite these problems, recent proteomics studies have identified novel proteins and have also led to a greater understanding of the “peptidome,” the small peptides of many proteins found in the circulation and in other compartments that result from protease digestion that arise in inflammatory and neoplastic states (199).

6 Conclusion In conclusion, the molecular profiles of pancreatic cancer continue to be elucidated. In particular, comprehensive sequencing of the pancreatic cancer genome and continued characterization of epigenetic and proteomic profiles of pancreatic cancer is helping scientists to better understand the biology of this devastating disease. Perhaps more importantly, these discoveries are forming the basis of a new generation of studies evaluating molecular diagnostic markers of pancreatic neoplasia. Accurate markers of preinvasive pancreatic neoplasms can be used to help predict future cancer risk among individuals undergoing pancreatic screening. It is also likely that we will also see advances in our understanding and management of familial pancreatic cancer led by the discovery of additional genes responsible for pancreatic cancer susceptibility. Such discoveries will provide opportunities for genetic counseling and screening. Ultimately, it is expected that the discovery of better molecular markers of pancreatic cancer will be useful to help decide whether a patient’s cancer is likely to respond to the novel therapies. Acknowledgment This work was supported by the National Cancer Institute grant (CA90709, CA120432, CA62924), and the Michael Rolfe Foundation.

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Section III

Pancreatic Cancer Staging

Chapter 13

Imaging of Pancreatic Adenocarcinoma Haydee Ojeda-Fournier and K. Ann Choe

Imaging plays a crucial role in the diagnosis, staging, and follow-up of pancreatic adenocarcinoma. In addition to identifying the primary tumor, the goals of imaging in pancreatic cancer include assessment of local and regional invasion, evaluation of lymph nodes and vascular structures, and evaluation for possible metastatic disease. Furthermore, imaging assessment identifies those patients who are candidates for resection—the only known therapy associated with longterm survival—and spares others from unnecessary surgical intervention. There is no indication for plain film evaluation and limited value in using ultrasonography (US) in the evaluation of pancreatic cancer. Because of its invasive nature, angiography has fallen out of favor in the evaluation of pancreatic adenocarcinoma. Cross-sectional imaging, including computed tomography (CT) and MRI, however, meet the stated goals in the assessment of pancreatic cancer. Magnetic resonance cholangiopancreatography (MRCP) can also add valuable information in specific cases. Other important modalities that utilize imaging include EUS, endoscopic retrograde pancreatography (ERCP), and PET are discussed elsewhere in this text.

Ultrasonography (US) Indication Sonography is widely available, relatively inexpensive, and noninvasive. It is often used for initial screenings of nonspecific abdominal complaints and evaluation of jaundice. Additional advantages of US include portability, ability to evaluate in real-time and the role it plays in image-guided procedures and intraoperative evaluations. In general, disadvantages of sonography include operator dependence, artifacts—particularly those caused by bowel gas—and limitations caused by patients’ body habitus. Because of the location of the pancreas in the retroperitoneum and bowel, neighboring the head and tail of the pancreas, transabdominal US evaluation is inherently limited. The gastric body is anterior to the tail of the pancreas, while A.M. Lowy et al. (eds.) Pancreatic Cancer. doi: 10.1007/978-0-387-69252-4, © Springer Science + Business Media, LLC 2008

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the duodenal loop surrounds the pancreatic head and jejunal loops and splenic flexure are anterior to the tail of the pancreas. As an initial screening study for nonspecific abdominal complaints, one of the best utilizations of US is in the evaluation of liver metastasis and assessment of the common bile and pancreatic ducts. The use of US intraoperatively—either laparoscopically or during a laparotomy—has been described as an additional tool in the evaluation of tumor respectability (1).

Technique The pancreas is evaluated as part of the general abdominal examination. It is best visualized after a 4- to 6-hour fast. This allows for decreased gaseous distention and for the gallbladder and biliary system to distend. Transverse and sagittal midline views, at a slight oblique angle below the level of the xiphoid, provide the best views of the pancreas transabdominally. A curved array detector with lower frequency is utilized to allow evaluation of deeper structures. In the 1990’s, laparoscopic US was reported as an effective tool used for assessing resectability with a reported accuracy of 94% (2). Intraoperative sonography has also been used and reported to be useful in the assessment of vascular invasion. In the series by Taylor et al. laparoscopic US prevented unnecessary additional surgery in 53% of their patients. With the new multidetector CT (MDCT) and the use of multiphasic contrast-enhanced protocols, however, the information previously provided by either laparoscopic or intraoperative US is often obsolete. The use of contrast-enhanced US is also reported in the literature (3) as a labor intensive experimental technique that may prove to be of use in the future for the evaluation of small pancreatic tumors; however, US contrast is not available for use in the United States. Again, the new generation CT scanners are superior, and thinner slice protocols can detect small tumors. In a meta-analysis comparing US, CT, and MRI, Bipat and colleagues estimated a sensitivity of 76% and a specificity of 75% for the sonographic diagnosis of pancreatic cancer and for resectability sensitivity and specificity of 83% and of 63%, respectively (4). Sonography had the lowest sensitivity and specificity as compared to the other modalities evaluated. Even more recent reports of better staging of resectability by intraoperative US over preoperative CT imaging are flawed by the use of conventional CT data (1).

Findings Characteristic findings of pancreatic cancer during sonography are a hypoechoic mass with ill-defined margins. Unfortunately this is indistinguishable from the sonographic appearance of focal pancreatitis. Associated findings include the presence of dilated common bile and pancreatic ducts (the so called double-duct sign indicating a tumor in the pancreatic head) (see Fig. 13.1), and atrophy of the pancreas. Assessment of lymph node involvement

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Fig. 13.1 Double duct sign seen by ultrasound. Note marked dilation of the common bile duct. The pancreatic duct which is usually not seen at the time of ultrasound is markedly dilated

by US, as with other modalities, is dependent on size criteria as well as the loss of normal morphologic appearance of the node, including echogenic hilum and smooth hypoechoic cortex. Micrometastasis to the lymph nodes cannot be excluded by the absence of these findings. Evaluation for direct invasion of adjacent organs can be limited by US secondary to location and bowel gas obscuration. Unexplained ascites should raise the suspicion of peritoneal surface metastasis, which is usually too small to resolve by US and other modalities. Evaluation of vessel invasion is better performed by CT. However, although not widely used, power Doppler and color Doppler assessment can prove useful in the assessment of the portal vein, celiac, common hepatic, and superior mesenteric arteries (5 -6 ).

Computed Tomography (CT) Indication CT is widely available as a screening tool for nonspecific abdominal complaints. For screening, a 5 mm slice thickness is obtained from the base of the lungs through the pelvis, usually with intravenous contrast during the portal venous phase. With the new helical multislice scanners, single breath hold images of the abdomen can be obtained in seconds. In this manner, CT screening can be used in those patients presenting with painless jaundice, abnormal LFT’s, abdominal pain,

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weight loss, and other nonspecific complaints that may be associated with pancreatic malignancy (7). Findings such as pancreas mass, common bile and pancreatic duct dilation, pancreas atrophy, and abnormalities in the contour of the pancreas should raise the suspicion of pancreatic adenocarcinoma. Suspicion of pancreatic pathology would then lead to targeted study with specialized protocols that can be applied for diagnosis, preoperative assessment of respectability, and post-operative follow-up of pancreatic adenocarcinoma. CT can also be used to guide procedures sometimes needed in the management of complications from the treatment of pancreatic cancer, including anastomotic leaks or abscess formation. In a recent prospective study of preoperative staging and tumor resectability assessment in pancreatic cancer, Soriano and colleagues found CT to have the highest accuracy in assessing the extent of disease, local regional extension, vascular invasion, distant metastases, TNM stage, and resectability, as compared to EUS, US, MRI, and angiography (8). In this study, EUS demonstrated a higher accuracy in assessing tumor size and regional lymph node involvement. Dedicated pancreas CT protocol thus is considered by most to be the study of choice in the initial assessment of pancreatic cancer. CT is widely available, requires little patient preparation, and can be performed in seconds. Abnormal renal function or a previous history of allergic reactions to contrast administration are the major contraindications to performing the contrast-enhanced CT.

Technique The development of pancreas-specific imaging protocols add specificity to the evaluation of pancreatic cancer and need to be distinguished from CT screening. Table 13.1 outlines the protocols utilized at our institution with the 16- and 64-slice multidetector row scanners. Utilization of a 64-slice scanner adds the capability of isinotropic imaging—the creation of multiplanar reformatted views from the raw data obtained. The coronal reformatted images are particularly helpful in assessing the cranio-caudal extent of lesions (see Fig. 13.2). In addition, a variety of other Table 13.1 Multiphasic pancreas CT protocol Patient position: Landmarks: Oral contrast: IV contrast: Amount: Rate: Injection delay: Pitch: Slice thickness: Interval: Reconstruction:

Supine Xiphoid to iliac crest Water Isoview 150cc 2cc/sec 40, 70, and 90 seconds 1 5mm 5mm 2.5 (through pancreas), 5, 5mm

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Fig. 13.2 Coronal reformatted CT image demonstrating cranio-caudal extent of pancreatic head mass, as well as invasion of the superior mesenteric vein

post-processing, 3D-imaging capabilities are available that can be applied in specific instances. These advanced technologies include maximum intensity projection (MIP), minimum intensity projection (MIN), volume rendering, and curved reformatting. These techniques are reportedly useful in problem-solving and in providing additional information to clinicians (9-11). MIP is useful in evaluating vessels, and provides a view of the highest-intensity pixels in the data. MIN images utilize the lowest density, and are helpful in evaluating fluid-filled structures such as the pancreatic and common bile ducts. Finally, a curved reformatted image is a technique in which the length of an irregular/curving structure can be “stretched” in a plane for better evaluation. Curved planar reformations can depict the degree of circumferential vascular involvement of the tumor—an important criterion in determining unresectability. In a study by Prokesch and colleges, however, curved planar reformations proved to be equivalent to transverse images in the assessment of tumor involvement along the vessel (10). Although all of these techniques are widely available, they require additional time to process and are often not necessary. At our institution, we reserve the use of these techniques for specific problemsolving situations in which additional imaging helps clarifies specific questions. The administration of intravenous contrast is of vital importance in the assessment of malignancy, and is optimized to differentiate tissue characteristics. Intravenous contrast is administered as a rapid bolus with a power injector at a rate of 3 to 5 mL/sec. Multiphasic contrast imaging is the accepted modality in evaluating pancreatic adenocarcinoma, although there is no consensus in the number of phases to be obtained (12-15). Specifically, Imbriaco and colleagues found no difference in the accuracy of single-phase versus dual-phase in resectability utilizing the helical

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CT technique (12). This group has further analyzed the utility of single-phase scanning utilizing MDCT and a delay of 60 seconds. In this study, scanning was performed in a caudal-to-cranial direction and again demonstrated single-phase scanning to be no different than multiphasic evaluation (16). In multiphasic evaluation, a noncontrast series is obtained for localization of the pancreas, which is also useful in evaluation of pancreatic calcifications, although calcifications are not a characteristic of adenocarcinoma. The arterial phase (20-30 seconds after the start of contrast infusion) has been reported to be unnecessary in the detection of pancreas adenocarcinoma (13), but it is useful in the assessment of arterial anatomy. The pancreatic phase ranges from 40-50 seconds after the initiation of contrast infusion, and is the phase with the greatest tumor-to-gland attenuation difference. During this phase, thin-cut axial images of the pancreas are obtained. Finally, the hepatic phase (60-70 second delay) is obtained throughout the entire abdomen and is necessary in the evaluation of metastatic disease of the liver. Fletcher et al. also demonstrated improved sensitivity for vascular invasion during the hepatic phase; however, there were also false positive cases (13). More recently, the combination of multiphasic CT with dedicated thin-cut slices through the pancreas has been shown to be sensitive and highly specific in the detection of pancreas adenocarcinoma, with improvement in detection rate of tumors smaller than 2 cm (15).

Findings Classic appearance of pancreatic adenocarcinoma is that of a low-density mass in the pancreas (see Fig. 13.3). Atrophy of the pancreatic gland, a change in the caliber of the pancreatic and common bile ducts, and contour deformity of the surface of the pancreas are additional findings in CT imaging (see Fig. 13.4). The attenuation appearance of the tumor varies and has been noted to be hypo-attenuating in 83% in one series, and iso-attenuating in 42% in other series (11). Most of the tumors (80%) are encountered in the head of the pancreas, and those in the body and tail present in a delayed fashion and have a worse prognosis (7, 11, 17). The main differential consideration is chronic pancreatitis, which has several imaging characteristics similar to pancreatic cancer, including atrophy of the gland, pancreatic duct dilation, and fibrotic-type low-density mass (7, 11, 18, 19). Chronic pancreatitis is often associated with coarse pancreas calcification, which is not a feature of pancreas cancer. Once adenocarcinoma of the pancreas is identified, assessment of resectability becomes the next crucial step. It is critical to identify those patients that are not resectable to spare them the morbidity and mortality risk associated with surgical intervention. Table 13.2 summarizes the findings of unresectability. Patients are considered unresectable if there is metastatic disease, local invasion, or involvement of major vessels including superior mesenteric artery, celiac axis. Limited involvement of the superior mesenteric vein/portal vein or hepatic artery may be amenable to resection and surgical reconstruction, therefore the extent of involvement needs to be

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Fig. 13.3 Classic CT appearance of pancreatic cancer, demonstrating a low-density lesion in the head of the pancreas. Note that the superior mesenteric artery and vein have fatty tissue circumferentially and are clear of invasive disease

Fig. 13.4 Axial CT image at the level of the pancreas demonstrating pancreatic atrophy, pancreatic and common bile duct dilation, and intrahepatic biliary ductal dilation

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H. Ojeda-Fournier, K.A. Choe Table 13.2 CT criteria for unresectability Distant metastasis Extensive Local invasion Peritoneal involvement Malignant ascites/pleural effusion Regional lymphadenopathy Vessel encasement greater than 50%, occlusion

Fig. 13.5A and B Advanced pancreatic cancer. In Fig. 13.5A, there are liver metastases, peritoneal implants, and malignant ascites. In Fig. 13.5B, at the level of the umbilicus, the Sister Mary Joseph lymph node is visualized. In addition, malignant ascites and peritoneal disease is also noted

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carefully described along with the presence or absence of tumor associated thrombus (6, 11, 17). Metastatic disease typically involves the liver and other distant organs, large volume malignant ascites, and peritoneal surfaces (see Fig. 13.5A and B). Classic appearance of liver metastasis is that of a ring-enhancing lesion or lowattenuation lesion in the liver. CT is generally limited in its ability to resolve small peritoneal surface metastasis, small liver metastasis, and micrometastasis to lymph nodes. The stomach, duodenum, and bile ducts are the organs most often involved with local invasion (20). When the tumor is very advanced, invasion of the colon, spleen, kidneys, and spine is possible (ref). An equivocal finding for unresectability includes masses greater than 3cm (11). Once the presence of distant metastatic disease and local invasion is excluded, it becomes essential to identify vascular involvement as another sign of nonresectability. Vessel involvement—including occlusion, deformation, and encasement—is considered a sign of unresectability (see Figs. 13.6, 13.7A, and 13.7B). Invasion or encasement of the portal vein is not an absolute contraindication to resection, however, as long as less than 2 cm of the vein is involved. There are surgical techniques in which vein grafts and/or patches are utilized in an attempt to achieve curative resection (21). The percent of vascular encasement by the tumor has been shown to be a useful predictor of resectability (20). Lu et al. developed a grading system for tumor involvement based on circumferential involvement, and concluded that tumor with greater than 50% circumferential involvement was a highly specific predictor of unresectability (22). Lu and colleagues consider the following five vessels as critical for nonresectability based on their surgical colleges’ operative procedure—celiac artery, hepatic artery, superior mesenteric artery, portal vein, and superior mesenteric vein. The grading

Fig. 13.6 Approximately 50% encasement of the superior vena cava is noted

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Fig. 13.7A and B: Circumferential encasement of the superior mesenteric artery. In Fig. 13.7A, the proximal SMA is visualized as involved to the level of the origin from the aorta. At a more caudal level, the pancreatic head mass is seen circumferentially encasing the SMA 209 210 211 212 213 214

scale that was developed considers a quartile of contiguous involvement of tumor along the vessel as outlined in Table 13.3. These observations have been further validated by others (20), and we include observations regarding vessel involvement in the standard evaluation of pancreatic adenocarcinoma. Staging of pancreatic cancer is based on the American Joint Committee on Cancer (AJCC) TNM staging system. Table 13.4 outlines the most recent updated

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Table 13.3 Vessel encasement, adapted from Lu (22) Grade 0 Grade 1 Grade 2 Grade 3 Grade 4

No tumor Less than 25% 25-50% 50-75% Greater than 75%

Table 13.4 TNM-staging exocrine pancreas Primary tumor T0 Tis T1 T2 T3 T4 Tx Regional lymph nodes N0 N1 Nx Distant metastasis M0 M1 Mx

No evidence of primary tumor Carcinoma in situ Tumor limited to pancreas, 2cm Tumor extends beyond the pancreas but without involvement of the celiac axis or the superior mesenteric artery Tumor involves the celiac axis or SMA Primary tumor cannot be assessed No regional lymph node metastases Regional lymph node metastases Cannot be assessed No distant metastasis Distant metastasis Cannot be assessed

TNM staging of pancreas cancer definition (23). The use of CT in predicting TNM staging was reported by Zeman and colleagues as effective for tumor and metastasis portion of the TNM staging, but not for the detection of lymph node metastasis (24). In that study, the previous TNM staging was utilized, as well as helical CT. As far as we know, this study has not been replicated utilizing the updated TNM definition and MDCT; however, one can speculate that assessment of lymph node involvement—particularly micrometastasis—will continue to be poor. This assessment may prove to be may be superior by PET/CT.

Magnetic Resonance Imaging (MRI) Indication Because of limited availability, cost, and time requirements, MRI has not surpassed CT as the modality of choice in the evaluation of pancreatic adenocarcinoma. Although it has better soft tissue resolution and reports of equal-to-improved

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sensitivity in the assessment of pancreatic cancer (25), MRI is reserved for those patients with an allergy to iodinated contrast and abnormal renal function, and for those patients in which MRCP is being performed concurrently. Because of the difference in technique and indication, MRCP is described separately. The main advantages of MRI are the increased soft tissue resolution and the ability to image in multiple planes, although this last capability is rivaled by MDCT. Another advantage of MRI includes the ability to perform angiography—either with gad enhancement or utilizing a 2D time-of-flight technique—without intravenous contrast administration. A disadvantage of MRI includes motion degradation secondary to the length of time in acquiring individual sequences, however this is less of an issue with newer, faster sequences utilizing breath hold techniques. Patient factors are also a significant drawback to utilization of MRI and include claustrophobia and patient’s body habitus. Claustrophobia can be decreased by the administration of mild sedatives. A scanner table weight limit of 300 lbs precludes the MR imaging of obese patients. The overall accuracy in determining tumor nonresectability, sensitivity, and specificity for MRI was reported to be 85%, 69%, and 95%, respectively, in a prospective study by Lopez Hanninen et al. (26). There are conflicting reports in the literature regarding whether CT or MRI is more accurate in the detection of pancreatic carcinoma (27). Given the wide availability of CT, the short time required to image, and little patient preparation, we consider CT superior but do acknowledge MRI’s better soft tissue contrast.

Technique MRI is contraindicated in patients with indwelling cardiac pacemakers, cochlear implants, certain types of aneurysm clips, and a variety of metals that are susceptible to high magnetic fields. A variety of imaging protocols can be used to evaluate the pancreas and upper abdomen as part of staging with MRI for pancreatic cancer. These protocols vary depending on the equipment being utilized and radiologists’ preferences. The sequences most often used include T1 and T1 fat-suppressed sequences, dynamic T1 post contrast sequences, and T2 sequences. The traditional intravenous contrast utilized in MRI is gadolinium, however the new contrast agent mangafodipir trisodium is reported to be taken up by hepatocytes and normal pancreas parenchyma, thus improving lesion detection in both the liver and pancreas (27-28).

Findings Findings of pancreatic adenocarcinoma are similar to those of CT and include the presence of a pancreatic mass, contour deformity of the surface of the gland,

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atrophy of the pancreatic gland, and double duct signs. A normal pancreas is lightly hyperintense to the liver in T1 sequences. Pancreatic tumors have lower signal intensity than normal pancreatic parenchyma on both T1 pre- and postgadolinium sequences. As with CT, differentiating chronic pancreatitis from pancreatic adenocarcinoma is problematic because chronic pancreatitis is also hypointense in T1 sequences (25, 29). Staging of pancreatic cancer with MRI is no different than with CT, and the same AJCC TNM staging definitions are applied. Likewise, assessment of major vessel encasement by a tumor is no different than the protocol applied by CT.

Magnetic Resonance Cholangiopancreatography (MRCP) Indication MRCP is a noninvasive alternative to ERCP, but as such it lacks the capability for cytology and histology analysis. MRCP images the biliary tract and pancreatic duct using water signals—i.e., T2 signal. One of the main advantages of MRCP over ERCP is the fact that there are no complications associated with this procedure—in addition, operator dependence is less of an issue. Ducts distal to areas of obstruction can also be visualized by MRCP, while this is often not possible with ERCP.

Technique Water sensitive sequences are prescribed to image the biliary system, hepatic ducts, and pancreatic duct in a noninvasive manner. The study is tailored to each individual patient, and the radiologist at our institution is frequently present to protocol and assess image acquisition.

Findings Dilation of the pancreatic duct by obstructing lesions is the main imaging finding of MRCP. A useful sign for distinguishing inflammatory process from carcinoma is the “duct penetrating sign,” which is seen more often in pancreatitis (85%)—in comparison with pancreatic cancer (4%) (25). The duct penetration sign is defined as the main pancreatic duct—either normal in caliber or smoothly stenotic— “penetrating” a pancreatic mass (30).

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Resectable

Screening CT or US Endoscopic US

Nonresectable palliation

Equivocal CT findings

Identification of metastasis

Non-resectable palliation

MRI if allergic to IV contrast

Dedicated CT pancreas protocol

Suspect metastasis

PET CT

Identification of metastasis

No metastasis

Non-resectable palliation

Resectable

Fig. 13.8 Suggested algorithm for imaging of suspected pancreatic adenocarcinoma

Conclusion Imaging plays an essential role in the diagnosis, preoperative assessment, and follow-up of pancreatic adenocarcinoma. A number of imaging modalities are available of which CT—because of its availability, relatively low cost, and speed—is regarded as the modality of choice. Unresectability of a tumor has been shown to

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be accurate using the TNM criteria of distant disease and major vessel involvement. Identification of small liver lesions, peritoneal disease, and lymph node metastasis, however, remains a primary drawback in the accurate assessment of resectability. Differentiation of tumors from chronic pancreatitis also remains a concern. Continued advances in imaging protocols and techniques are to be expected with the goals of improvement in assessing resectability and tumor staging.

References 1. Taylor AM, Roberts SA, Manson JM 2001, Experience with laparoscopic ultrasonography for defining tumour resectability in carcinoma of the pancreatic head and periampullary region. Brit J of Surg 88(8):1077–1083 2. Long EE et al. 2005, Computed tomography, endoscopic, laparoscopic, and intra-operative sonography for assessing resectability of pancreatic cancer. Surg Onc 14(2):105–113 3. Oshikawa O et al. 2002, Dynamic sonography of pancreatic tumors: comparison with dynamic CT. AJR. Am J of Roentgenology 178(5):1133–1137 4. Bipat S et al. 2005, Ultrasonography, computed tomography and magnetic resonance imaging for diagnosis and determining resectability of pancreatic adenocarcinoma: a meta-analysis. J of Comp Asst Tomog 29(4):438–445 5. Kaneko T et al. 2002, Power Doppler ultrasonography for the assessment of vascular invasion by pancreatic cancer. Pancreatology: Off J of the Intl Assoc of Pancreatology (IAP) 2(1):61–68 6. Clarke DL et al. 2003, Preoperative imaging of pancreatic cancer: a management-oriented approach. J of the Am Coll of Surg 196(1):119–129. 7. Katz MH et al. 2005, An evidence-based approach to the diagnosis and staging of pancreatic cancer. Pancreatology: Off J of the Intl Assoc of Pancreatology (IAP) 5(6):576–590 8. Soriano A et al. 2004, Preoperative staging and tumor resectability assessment of pancreatic cancer: prospective study comparing endoscopic ultrasonography, helical computed tomography, magnetic resonance imaging, and angiography. Am J of Gastroenterology 99(3):492–501 9. Tamm E, Charnsangavej C, Szklaruk J 2001, Advanced 3-D imaging for the evaluation of pancreatic cancer with multidetector CT. Intl J of Gastro Cancer 30(1-2):65–71. 10. Prokesch RW et al. 2002, Local staging of pancreatic carcinoma with multi-detector row CT: use of curved planar reformations initial experience. Radiology 225(3):759–765. 11. Smith SL, Rajan PS 2004, Imaging of pancreatic adenocarcinoma with emphasis on multidetector CT. Clin Radiol 59(1):26–38 12. Imbriaco M et al. 2002, Dual-phase versus single-phase helical CT to detect and assess resectability of pancreatic carcinoma. Am J of Roentgenology (AJR) 178(6):1473–1479 13. Fletcher JG et al. 2003, Pancreatic malignancy: value of arterial, pancreatic, and hepatic phase imaging with multi-detector row CT. Radiology 229(1):81–90 14. Valls C et al. 2002, Dual-phase helical CT of pancreatic adenocarcinoma: assessment of resectability before surgeryAm J of Roentgenology (AJR) 178: p. (4)821–6. 15. Bronstein YL et al. 2004, Detection of small pancreatic tumors with multiphasic helical CT. AJR. Am J of Roentgenology (AJR) 182(3):619–623 16. Imbriaco M et al. 2005, Value of the single-phase technique in MDCT assessment of pancreatic tumors. Am J of Roentgenology (AJR) 184(4):1111–1117 17. Balci NC, Semelka RC 2001, Radiologic diagnosis and staging of pancreatic ductal adenocarcinoma. Euro J of Radiol 38(2):105–112 18. Tamm E, Charnsangavej C 2001, Pancreatic cancer: current concepts in imaging for diagnosis and staging. Cancer J 7(4):298–311

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19. Leung T-K et al. 2005, Difficulty with diagnosis of malignant pancreatic neoplasms coexisting with chronic pancreatitis. World J of Gastroent (WJG) 11(32):5075–5078 20. O’Malley ME et al. 1999, Adenocarcinoma of the head of the pancreas: Determination of surgical unresectability with thin-section pancreatic-phase helical CT. Am J of Roentgenology (AJR) 173:1513–1518 21. Obuz F et al. 2001, Pancreatic adenocarcinoma: detection and staging with dynamic MR imaging. Eur J of Radiol 38(2):146–150 22. Lu DSK et al. 1997, Local staging of pancreatic cancer: criteria for unresectability of major vessels as revealed by pancreatic-phase, thin-section helical CT. Am J of Roentgenology (AJR) 168:1439–1443 23. Exocrine pancreas. In: American Joint Committee on Cancer: AJCC Cancer Staging Manual, 6th ed. New York, NY: Springer, 2002:157-164 24. Zeman RK et al. 1997, TNM Staging of Pancreatic Carcinoma Using Helical CT. Am J of Roentgenology (AJR) 169:459–464 25. Fayad LM, Mitchell DG 2001, Magnetic resonance imaging of pancreatic adenocarcinoma. Int J of Gastroint Cancer 30(1-2):19–25 26. Lopez Hanninen E et al. 2002, Prospective evaluation of pancreatic tumors: accuracy of MR imaging with MR cholangiopancreatography and MR angiography. Radiology 224(1):34–41 27. Ramsay D et al. 2004, Identification and staging of pancreatic tumours using computed tomography, endoscopic ultrasound and mangafodipir trisodium-enhanced magnetic resonance imaging. Australasian Radiol 48(2):154–161 28. Schima W et al. 2002, Diagnosis and staging of pancreatic cancer: comparison of mangafodipir trisodium-enhanced MR imaging and contrast-enhanced helical hydro-CT. Am J of Roentgenology (AJR) 179(3):717–24 29. Fischer U et al. 2002, Preoperative local MRI-staging of patients with a suspected pancreatic mass. Euro Radiol 12(2):296–303 30. Ichikawa T et al. 2001, Duct-penetrating sign at MRCP: usefulness for differentiating inflammatory pancreatic mass from pancreatic carcinomas. Radiology 221(1):107–116

Chapter 14

The Role of PET Scanning in Pancreatic Cancer M. Fernandez-Ulloa

1 Introduction For many years computed tomography (CT) has been the principal imaging modality used for diagnosis and staging of patients with pancreatic cancer. Currently, contrast enhanced multidetector CT (ce MDCT), remains the most commonly used imaging study in the initial evaluation of these patients. Depending on clinical practices and preferences, other modalities, such as magnetic resonance imaging (MRI) and endoscopic ultrasonography (EUS), are also utilized in the diagnosis and staging of pancreatic carcinoma (1, 2). The use of positron emission tomography (PET) in routine oncology practice has accelerated significantly in the past decade. Oncologic PET imaging is mainly performed with F18-FDG (FDG), a glucose analog that accumulates in cancer cells. This modality is under investigation in the evaluation of patients with pancreatic carcinoma as a diagnostic tool to better define extent of disease and guide treatment. Specifically, the ability of FDG-PET to find metastasis to the liver and elsewhere may be of great value in the management of patients with pancreatic cancer.

2 Principles of Cancer Imaging with F18-FDG Fluorine-18 belongs to the family of positron emitting radioisotopes. Unstable neutron deficient nuclei in these radioisotopes emit positively charged electrons. These positrons traverse varying distances in tissues depending on their energy but eventually collide with electrons surrounding nuclei and are annihilated. The result of this annihilation is the emission of two gamma rays at a 180-degree angle. This provides the basis for PET imaging. One of the main characteristics of PET is its ability to provide spatial information used for processing and image display in tomographic formats, that is, axial, coronal, and sagittal planes. Other positron emitters include carbon-11, nitrogen-13, and oxygen-15. As opposed to F-18, which has a half-life of 120 minutes, these three have very short half-lives, which A.M. Lowy et al. (eds.) Pancreatic Cancer. doi: 10.1007/978-0-387-69252-4, © Springer Science + Business Media, LLC 2008

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makes them impractical for clinical use. Thus, F-18 is currently the most important PET imaging radioisotope. Substitution of F-18 for the hydroxyl in the carbon 2 of the glucose yields the glucose analog 2-(fluorine-18) fluoro-2-deoxy-D-glucose, or F18-FDG. As a result of this modification to the chemical structure, the metabolic characteristics of F18-FDG differ from those of glucose. Accordingly, F18- FDG is transported into the cells via glucose transporters (Glut) and phosphorylated by the hexokinase enzyme to FDG-6-phosphate, but cannot be metabolized further and is essentially trapped intracellularly since G-6-phosphatase is low in these cells. The unique kinetics of F18-FDG allow for imaging of tumors with PET. The use of F18-FDG for imaging of cancer emerged from the long known concept that cancer cells exist in a hypermetabolic state and concentrate glucose at high levels. Increased accumulation of the glucose analog F18-FDG in cancer cells is caused, to a significant extent, by oncogene-induced increased glucose consumption and activation of glucose transporters (3). The uptake of FDG in tumors is also affected by multiple factors, some of which are not well understood. These include the number of viable cells, blood supply, presence of hypoxia, blood sugar and insulin levels, degree of expression of Glut at the cell membrane and the effect of therapeutic interventions such as chemotherapy and radiotherapy. One rationale for the use of FDG-PET imaging in pancreatic cancer rests on evidence of Glut-1 transporter over expression in pancreatic cancer specimens (4–6). The presence or absence of FDG accumulation in pancreatic tumors and other tissues can be assessed well with PET alone. However, since PET lacks the anatomic detail necessary for optimal characterization of normal tissue FDG uptake and precise localization of neoplastic lesions, PET-CT was introduced to overcome these limitations and has largely replaced PET. In these systems the PET and the CT are integrated into one single imaging devise, a design that offers several advantages. The ability to obtain the PET and CT data sets almost simultaneously allows for almost perfect fusion of the two images, resulting in precise localization of PET abnormalities on the anatomic CT images. Also normal accumulation of FDG in the bowel and other intra-abdominal organs can easily be determined, which minimizes false-positive and enhances specificity of the study. Similarly suspicious CT lesions can be evaluated more precisely for the presence of FDG uptake within them. As in any nuclear medicine technique, the effect of tissue attenuation of gamma photons is an important factor that causes loss of resolution and image detail. This problem is dealt with by applying the attenuation maps from the CT data to the PET data during image processing. It should be emphasized that absolute correspondence of the PET and CT images is not always possible, due to the fact that acquisition of the PET and CT data is not simultaneous. Thus, patient and organ movement during and between imaging sessions may result in varying degrees of image misregistration and suboptimal application of the CT derived soft tissue attenuation map to the PET data during image reconstruction. Several brands of PET and PET-CT imaging devices are currently in use, but the clinical imaging performance of these instruments is comparable. This likely will change as various new innovations to the equipment are introduced.

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The operation of PET-CT calls for proper tailoring imaging of protocols depending on the clinical situation. The CT portion of any PET-CT study may differ, depending on practice preferences and the clinical question. When the CT is used for anatomic correlation and attenuation correction purposes only, a nondiagnostic low-dose CT is obtained. Although detail and diagnostic content of these CTs are somewhat inferior, most CTs in current PET-CT systems are of very good quality. A diagnostic CT typically is performed with the aid of intravenous contrast media and occasionally oral contrast as well. Acquisition parameters for diagnostic CTs are also modified accordingly. For the PET images, patients are given the FDG intravenously approximately 1 hour before imaging. The imaging sequence starts with a “scout” CT, followed by the actual CT imaging session and ends with the PET imaging. Prior to the administration of the FDG patients fast for at least 6 hours and are required to avoid strenuous physical activities. The blood sugar ideally should be within normal limits, but levels of up to 150 mg may not affect the kinetics of FDG significantly. Administration of insulin should be avoided for at least 4 hours before the FDG injection. The use of PET-CT involves radiation exposure to patients. Since combined PET-CT systems are rapidly replacing the dedicated PET system, radiation exposure to patients from both the PET and CT needs to be considered. The effective radiation dose from a standard dose of FDG 370 MBq (10 mCi) is approximately 7 mSv. The addition of a low-dose CT study increases the total effective dose to about 25 mSv (7). Slightly higher radiation exposures are expected if a diagnostic CT study is obtained. As a comparison annual radiation exposure to the general public from background radiation is about 3–4 mSv, depending on geographic location. Thus, the risk-benefit ratio of PET-CT imaging is low and within acceptable levels considering the seriousness of pancreatic cancer.

3 Image Evaluation and Interpretation Examination of FDG images is performed visually and semiquantitatively from the standard uptake values (SUVs). Usually, the FDG-PET and CT set are evaluated separately followed by examination of the fused images to cross-correlate findings and abnormalities. Analysis of the pattern of FDG uptake when evaluating PET-CT images in patients with pancreatic masses helps determine their malignant or benign etiology. A well-circumscribed focal area of intense uptake is generally indicative of malignancy, whereas FDG uptake that is more diffuse and less intense is suggestive of chronic pancreatitis (8). In addition to the visual inspection of images, SUVs are used in an attempt to provide an objective parameter that estimates the degree of FDG uptake in lesions. SUVs represent a semiquantitative expression of the concentration of FDG in tumors calculated as follows:

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PET counts × calibration factor (mCi/g) SUV = --------------------------------------------------------------------------------------------------Dose of injected FDG (mCi)/body weight (g) or the percent of injected dose in the lesion normalized for body mass. To determine the PET counts a volumetric region of interest (ROI) is selected from the axial images encompassing the tumor area with the maximal metabolic activity. Usually two SUV determinations are generated; the average and the maximal. The maximal SUV looks at the highest level of FDG concentration within lesions. The average SUV expresses the differences of distribution of FDG within a tumor and takes into consideration the variation of FDG concentration throughout a lesion due to its heterogeneity of distribution within tumors. Factors that may actually decrease concentration of FDG within a tumor, such as necrosis and blood flow, may affect average SUV values. For this reason, use of maximal SUVs that express the maximal metabolic activity within a tumor is favored. SUVs at various cutoff levels have been proposed as discriminators between cancer and other non-neoplastic processes. There are limitations to the use of SUVs as discriminators, however, as they can be affected by many technical as well as physiologic factors. These include calibration of the PET instruments; selection of ROI, blood sugar level, time elapsed between injection of FDG and imaging, and heterogeneity of FDG uptake in tumors. The effect of these factors can be reduced by proper and detailed attention to patient preparation and imaging techniques. The main limitation, however, is the significant overlap of FDG uptake between cancer and other nonneoplastic processes such as nonspecific inflammation, infection, and granulomatous diseases. In regard to pancreatic cancer, the value of SUVs as tools for diagnosis and evaluation of these patients has not been established. SUVs were found helpful to differentiate pancreas cancer from chronic pancreatitis (8) and improve sensitivity for pancreas cancer detection compared with visual image inspection. In an effort to better refine the usefulness of SUVs, they have been combined with tracer retention indexes (RI). These methods compare the SUV values in tumors at two time (T1, T2) intervals, following the injection of the FDG as follows: SUV2-SUV1 RI = ----------------------- × 100% SUV1 A common practice is to obtain these SUVs at 1 and 2 hours after injection of FDG. As opposed to benign processes, cancer usually shows higher RIs (9). Disadvantages of this technique include the need for additional imaging of the patient, which makes it impractical in routine clinical practice. The usefulness of SUVs and retention indexes is further limited by the difficulty in defining precise cutoff values that provide the best discriminator function to separate cancer from benign diseases. Moreover, validated SUVs and RIs that differentiate malignant from benign pancreatic lesions that can be interchanged between institutions have not been established. While the implementation of these semiqualitative techniques in clinical practice will need further validation of its usefulness, they mainly remain an important tool of clinical research.

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4 Limitations of FDG-PET Imaging One of the main limitations of FDG imaging is its specificity for cancer. It is a wellknown fact that in addition to cancer, uptake of FDG is also observed in various inflammatory and granulomatous processes. Since F18-FDG commonly concentrates in the bowel as well in the kidneys and urinary tract, evaluation of these regions can be difficult. Physiologic accumulation of FDG in these organs also affects negatively the specificity of PET for cancer. The advent of PET-CT has minimized this problem significantly by reducing the number of false-positives caused by this physiologic FDG uptake. False-negative results can occur when lesions are 150 mg/dl.

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5 Value of FDG-PET Imaging for Detecting Primary Pancreatic Cancer FDG-PET for the diagnosis of pancreatic carcinoma has been explored in three main clinical situations: at the initial diagnosis in patients with suspicious masses, for differentiating benign pancreatic disease such as chronic pancreatitis from cancer, and in the evaluation of cystic lesions. The majority of pancreatic carcinomas present clinically at a relatively advanced stage and only about 20% of patients are found resectable at the time of diagnosis (16). Imaging with CT and MRI as well as EUS provides the more accurate noninvasive means for confirmation of diagnosis and staging prior to treatment. Use of FDG-PET for evaluation of the primary tumor and pancreatic masses is still controversial, given its limited sensitivity and specificity (17). It should be noted that most published studies report the use of dedicated PET systems. Only recently have new data on FDG imaging with combined PET-CT instruments begun to emerge. However, several studies suggest that FDG-PET may have utility in the diagnosis of cancer in patients with pancreatic masses (9, 10, 18, 19). A study examining the value of FDG-PET to diagnose cancer in 106 suspicious lesions reported a sensitivity and specificity of 85% and 84%, respectively (10). Additionally, comparisons with other diagnostic modalities including CT, EUS, and MRI have found FDG-PET of comparable accuracy (9, 18). In a study of 80 patients, sensitivity and specificity of FDG-PET to detect pancreatic cancer were 88% and 92%, respectively (20). A recent tabulated review of 387 patients demonstrated a weighted average sensitivity of 94% and specificity of 90% for detection of primary pancreatic cancer (21). Thus, there is emerging clinical evidence that FDG-PET is helpful in the evaluation of patients presenting with a pancreatic mass. The recent emergence of PET-CT has introduced new questions regarding the role and utility of FDG imaging in patients with pancreatic cancer. Because of the simultaneous whole body anatomic and metabolic information that can be obtained in one single imaging session, this technique has the potential to add significant diagnostic value to oncologic FDG imaging. A recent report on the use of digitally fused MDCT and FDG-PET images described an improvement in sensitivity and specificity, as well as positive and negative predictive values in the differential diagnoses between benign and malignant lesions as compared with PET alone (22). A more recent study comparing dual FDG-PET-CT with contrast-enhanced CT in the diagnosis of primary pancreatic carcinoma lesions found sensitivities of 89% and 93%, respectively (23). The specificity for PET-CT and contrast enhanced CT were 93% and 21%, respectively. The positive and negative predictive values for PET-CT were 91% and 64%, respectively. Thus, although in this study combined PET-CT appears to be sensitive and specific, its negative predictive value for detection of pancreatic cancer was low and therefore unreliable. The usefulness and value of combined PET-CT in the evaluation of primary pancreatic lesions is currently unknown; extensive clinical studies will be needed to confirm initial positive results. An important practical question now is how the use of PET-CT will impact

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the overall value of FDG imaging in difficult clinical situations. Given the usual late clinical presentation of pancreatic carcinoma, the main aim of diagnostic imaging is to separate those patients who have a chance at surgical care from those who do not. In this context PET or PET-CT may not reduce the need for invasive diagnostic procedures in patients with pancreatic masses. This is because from the clinician’s point of view, a positive FDG-PET only reaffirms the suspicion of cancer but does not necessarily obviate the need for a tissue diagnosis. Conversely, the imperfect negative predictive value of FDG-PET does not obviate the need for further invasive confirming diagnostic approaches. Similarly, PET is not well suited for evaluation of regional adenopathy and vascular invasion, which are an important determinants of resectability. Reported sensitivity and specificity in detecting lymph node disease is generally suboptimal because the metastases may be below PET resolution of 6–8 mm and varies widely between 46% and 71%, and 63% and 100%, respectively (24, 25). Thus, as discussed in the following, the main role of PET in pancreatic cancer is in the search for otherwise occult metastatic disease, which if present would change management.

6 Detection of Distant Metastases The presence of radiographically occult distant metastases is common at the time of diagnosis of pancreas cancer. This represents an important clinical problem since radical resections in patients with metastatic disease accomplish nothing and may even result in deleterious outcomes. FDG-PET imaging has been found to be accurate in the detection of hepatic and additional sites of distant metastatic disease (26–28). Although false-positive results may be caused by intrahepatic cholestasis, in some studies, imaging with FDG-PET has shown to be more accurate than CT for detection of metastases to the liver and can detect disease at other distant sites (29, 30) (Figs. 14.1 and 14.2). False-negatives are seen when tumors 18 mm PV/ SMV invasion survived >18 months after the resection. Involvement of the PV/ SMV by pancreatic carcinoma seems to be related to the extent of the disease and

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the PV/SMV involvement >18 mm is associated with a poor prognosis due to high rate of tumor positive margin even with radical operation.

9 Summary of EUS T Staging In a large retrospective analysis of studies of the accuracy of EUS and CT, DeWitt et al. have compared the two imaging modalities. It is uncertain whether computed tomography (CT) or endoscopic ultrasound (EUS) is superior for the detection, staging, and resectability of pancreatic cancer. A systematic literature review was carried out to determine which test is more accurate. They identified relevant studies from MEDLINE (1986–2004) and evaluated study quality, which was measured on the basis of guidelines for assessing studies of diagnostic tests. Quantitative outcomes data were abstracted from the studies. Eleven studies with 678 patients satisfied inclusion criteria. Nine studies assessed tumor detection, all of which concluded that the sensitivity of EUS was superior to CT. Four of five studies that assessed tumor staging accuracy and five of eight that assessed nodal staging accuracy concluded that EUS was superior to CT. Among the four studies that assessed resectability, two showed no difference between EUS and CT, and one favored each modality. Three of 11 studies met all but one of the quality criteria. The most important and frequent study limitations were lack of a consecutive series of patients and biased patient selection for surgery. Quantitative comparisons among studies were precluded by differences in tumor staging classifications, surgical selection, CT and EUS techniques, and reporting of operating characteristics. The published literature comparing EUS and CT for preoperative assessment of pancreatic cancer is heterogeneous in study design, quality, and results. All studies have methodologic limitations that potentially affect validity. Prospective studies with state-of-the-art imaging are needed to further define the role of each test.

10 EUS Lymph Node Staging EUS has generally excelled in its ability to detect malignant peripancreatic adenopathy. In a study of 160 patients, 78 had peripancreatic lymph nodes (49%), 25 had celiac lymph nodes (16%), and 14 patients had mediastinal lymph nodes (9%) that were suspicious for malignancy by morphologic criteria (19). In eight of 14 patients with suspicious mediastinal lymph nodes, FNA documented malignancy in 5%. Only one of these eight patients with malignant mediastinal adenopathy had other sites of documented distant metastases by CT and/or positron emission tomography scans. However, seven of eight patients had locally advanced cancers. Malignant mediastinal adenopathy is detected by staging EUS-FNA in 5% of patients with pancreaticobiliary cancer. Because of its important implications, endosonographers should routinely assess for malignant mediastinal adenopathy in

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patients who undergo staging EUS for pancreaticobiliary malignancy. Lymph node staging with EUS may provide even more important information than tumor staging (20). The outcome of pancreatic head cancer after surgical resection is still difficult to predict. In this study of 100 consecutive patients with invasive adenocarcinoma of the head of the pancreas, the results of surgical pathology were retrospectively analyzed to clarify the influence of clinicopathological factors. The overall 1-, 3-, and 5-year survival rates for the 100 patients with pancreatic head cancer were 55%, 16%, and 6%, respectively. Among the 16 clinicopathologic factors, nine were significantly associated with outcome in univariate analysis: tumor type (invasive ductal cancer), poor histologic differentiation, extrapancreatic plexus invasion, bile duct invasion, duodenal invasion, intrapancreatic nerve invasion, lymphatic invasion, venous invasion, and nodal involvement. Multivariate analysis confirmed that nodal involvement and extrapancreatic plexus invasion were significant independent factors for overall survival. Nodal involvement was the strongest predictor of poor survival after pancreatic resection for invasive adenocarcinoma of the head of the pancreas.

11 Conclusions Preoperative pancreatic cancer staging with EUS provides detailed images of the pancreatic mass and its relationship to the peripancreatic vasculature. The accuracy of EUS staging varies with instrumentation, endoscopists, and patient selection. EUS excels in lymph node staging through the use of EUS. Currently, EUS staging remains a tool for the evaluation of patients with known or suspected pancreatic cancer. EUS has become the preferred method to obtain tissue biopsies in patients with locally advanced disease.

References 1. Huang QJ, Xu Q, Wang XN, 2002, Spiral multi-phase CT in evaluating resectability of pancreatic carcinoma. Hepatobiliary Pancreat Dis Int 1(4):614–619. 2. Agarwal B, Abu-Hamda E, Molke KL, 2004, Endoscopic ultrasound-guided fine needle aspiration and multidetector spiral CT in the diagnosis of pancreatic cancer. Am J Gastroenterol 99(5):844–850. 3. Karmazanovsky G, Fedorov V, Kubyshkin V, 2005, Pancreatic head cancer: accuracy of CT in determination of resectability. Abdom Imaging 30(4):488–500. 4. Li H, Zeng MS, Zhou KR, 2005, Pancreatic adenocarcinoma: the different CT criteria for peripancreatic major arterial and venous invasion. J Comput Assist Tomogr 29(2):170–175. 5. Brugge WR, Lee MJ, Kelsey PB, 1996, The use of EUS to diagnose malignant portal venous system invasion by pancreatic cancer. Gastrointest Endosc 43(6):561–567. 6. Brugge WR. 1995, Pancreatic cancer staging. Endoscopic ultrasonography criteria for vascular invasion. Gastrointest Endosc Clin North Am 5(4):741–753. 7. Gress FG, Hawes RH, Savides TJ, 1999, Role of EUS in the preoperative staging of pancreatic cancer: a large single-center experience. Gastrointest Endosc 50(6):786–791.

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8. Rosch T, Dittler HJ, Strobel K, 2000, Endoscopic ultrasound criteria for vascular invasion in the staging of cancer of the head of the pancreas: a blind reevaluation of videotapes. Gastrointest Endosc 52(4):469–477. 9. Shoup M, Hodul P, Aranha GV, 2000, Defining a role for endoscopic ultrasound in staging periampullary tumors. Am J Surg 179(6):453–456. 10. Tierney WM, Francis IR, Eckhauser F, 2001, The accuracy of EUS and helical CT in the assessment of vascular invasion by peripapillary malignancy. Gastrointest Endosc 53(2):182–188. 11. Rivadeneira DE, Pochapin M, Grobmyer SR, 2003, Comparison of linear array endoscopic ultrasound and helical computed tomography for the staging of periampullary malignancies. Ann Surg Oncol 10(8):890–897. 12. Soriano A, Castells A, Ayuso C, 2004, Preoperative staging and tumor resectability assessment of pancreatic cancer: prospective study comparing endoscopic ultrasonography, helical computed tomography, magnetic resonance imaging, and angiography. Am J Gastroenterol 99(3):492–501. 13. Aslanian H, Salem R, Lee J, 2005, EUS diagnosis of vascular invasion in pancreatic cancer: surgical and histologic correlates. Am J Gastroenterol 100(6):1381–1385. 14. Bettini N, Moutardier V, Turrini O, 2005, Preoperative locoregional re-evaluation by endoscopic ultrasound in pancreatic ductal adenocarcinoma after neoadjuvant chemoradiation. Gastroenterol Clin Biol 29(6–7):659–663. 15. Fritscher-Ravens A, Knoefel WT, Krause C, 2005, Three-dimensional linear endoscopic ultrasound-feasibility of a novel technique applied for the detection of vessel involvement of pancreatic masses. Am J Gastroenterol 100(6):1296–1302. 16. Kobayashi A, Yamaguchi T, Ishihara T, 2005, Assessment of portal vein invasion in pancreatic cancer by fusion 3Dimensional ultrasonography. J Ultrasound Med 24(3):363–369. 17. Nakata B, Nishino H, Ogawa Y, 2005, Prognostic predictive value of endoscopic ultrasound findings for invasive ductal carcinomas of pancreatic head. Pancreas 30(3):200–205. 18. Tezel E, Kaneko T, Takeda S, 2005, Intraportal endovascular ultrasound for portal vein resection in pancreatic carcinoma. Hepatogastroenterology 52(61):237–242. 19. Agarwal B, Gogia S, Eloubeidi MA, 2005, Malignant mediastinal lymphadenopathy detected by staging EUS in patients with pancreaticobiliary cancer. Gastrointest Endosc 61(7):849–853. 20. Nakagohri T, Kinoshita T, Konishi M, 2006, Nodal involvement is strongest predictor of poor survival in patients with invasive adenocarcinoma of the head of the pancreas. Hepatogastroenterology 53(69):447–451.

Chapter 16

Laparoscopic Staging Matthew H. Katz, Abdool R. Moossa, and Michael Bouvet

1 Introduction Although pancreatic cancer currently ranks tenth in cancer incidence, it represents the fourth leading cause of cancer death in the United States among patients of both sexes (1). The therapeutic options available to individuals with this disease are primarily determined by disease stage. Patients with small, localized tumors are typically candidates for surgical resection by pancreaticoduodenectomy or distal pancreatectomy, which currently offer the only chance for longterm survival (2). In major, specialized centers, median survival of up to 20 months can now be achieved after complete tumor resection (3–5), with 5-year survival rates approaching 40–50% in selected patients (6, 7). Unfortunately, only 10–15% of patients with pancreatic cancer are found to have resectable tumors at the time of diagnosis, with the remainder harboring either locoregional extension or distant metastases (8) which preclude effective surgical therapy. For this overwhelming majority of patients presenting with advanced disease, treatment strategies are currently limited, and patient prognosis is therefore extremely poor—overall, individuals with advanced pancreatic malignancy have a 5-year survival rate less than 1%, with an overwhelming majority dead within 1 year of diagnosis (9, 10). Timely diagnosis and staging thus play a fundamental role in the evaluation of patients with pancreatic cancer. Two goals are paramount: (1) diagnosis of the disease early in its natural history, thereby providing patients an opportunity for curative surgical therapy; and (2) preoperative discrimination between those patients with early stage tumors in whom tumor resection is warranted, and those with unresectable disease, and consequently limited survival, in whom the morbidity and expense of major surgery should routinely be avoided. Until recently, tissue diagnosis, staging, and assessment of resectability were all accomplished during exploratory laparotomy. Over the past two decades, however, significant advances in diagnostic and staging technologies, such as computed tomography (CT), endoscopic ultrasonography (EUS), and staging laparoscopy, as well as the development of minimally invasive palliative strategies, have permitted a more selective approach to open surgery (11).

A.M. Lowy et al. (eds.) Pancreatic Cancer. doi: 10.1007/978-0-387-69252-4, © Springer Science + Business Media, LLC 2008

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First performed as early as 1911 at Johns Hopkins on a patient with pancreatic cancer (12), staging laparoscopy in the modern era began with Cuschieri (13), who described a series of 23 patients with pancreatic cancer in whom a laparoscopic method was found to be effective in both diagnosis of the disease and determination of operability. Since that time, the different techniques used as well as the potential indications for the procedure have been described and expanded upon in numerous reports. In aggregate, the available data clearly indicate that laparoscopic exploration can be used as a supplemental staging procedure to identify small peritoneal or liver metastases in certain patients in whom such advanced disease may have been missed using other preoperative imaging modalities. The technique can therefore obviate the need for open exploration in certain patients. Further reports have demonstrated the potential value of adjunctive methods including extended dissection, laparoscopic ultrasonography, and the analysis of laparoscopically obtained peritoneal cytologic samples to further enhance the utility of laparoscopic exploration. Controversy continues to exist, however, as to whether laparoscopic staging should be offered to all patients with the disease, or selectively to particular subgroups of patients. In our view, laparoscopic staging can be a safe and effective staging modality that should be offered selectively based on the likelihood that it will influence management in a given clinical scenario. This chapter reviews the current evidence supporting the use of staging laparoscopy in patients with a diagnosis of pancreatic cancer, and discusses its role in the algorithm we use to both stage and treat patients with this devastating disease.

2 American Joint Committee on Cancer Staging System for Pancreatic Cancer Recently updated, the American Joint Committee on Cancer (AJCC) TNM staging system is used for the uniform staging of pancreatic cancer (Table 16.1) (14) and should be well understood prior to analyzing the efficacy of any staging modality. In the AJCC system, disease stage is determined by three factors: (1) the size of the primary tumor and its relationship to the celiac axis and superior mesenteric artery (T); (2) the presence or absence of regional lymph node involvement (N); and (3) the presence or absence of distant metastases (M). Tumors diagnosed in stage I are small and localized to the pancreas, and are therefore routinely resectable. Stage II disease is characterized by a primary tumor that extends into adjacent organs or involves regional lymph nodes, without distant metastases or invasion into the celiac trunk or superior mesenteric artery. Such tumors, even those with isolated portal or superior mesenteric vein involvement, are also often resectable. Patients with stage III disease have locally advanced disease that involves the major arteries, and those with stage IV tumors are found to have distant metastases at the time of diagnosis. Neither group is eligible for surgical resection. Instead, patients with stage III disease are often enrolled in clinical trials utilizing chemoradiation protocols designed to achieve locoregional control; patients with stage IV disease typically do not receive radiation but may be offered palliative chemotherapy. Adjunctive procedures, such

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Table 16.1A TNM definitions for the staging of pancreatic cancer Primary tumor (T) TX Cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ T1 Tumor confined to pancreas, ≤2 cm in diameter T2 Tumor confined to pancreas, >2 cm in diameter T3 Extrapancreatic extension, no celiac axis or SMA involvement T4 Involvement of celiac axis or SMA Regional lymph nodes (N) NX N0 N1

Cannot be assessed Regional lymph node metastases absent Regional lymph node metastases present

Distant metastases (M) MX M0 M1

Cannot be assessed Distant metastases absent Distant metastases present

SMA, superior mesenteric artery. Table 16.1B AJCC TNM staging of pancreatic cancer Stage 0

Tis, N0, M0

Stage I Ia Ib

T1, N0, M0 T2, N0, M0

Stage II IIa IIb

T3, N0, M0 T1-3, N1, M0

Stage III

T4, N0-1, M0

Stage IV

T1-4, T0-1, M1

as surgical biliary-enteric bypass or endobiliary stenting, may also be employed to palliate symptoms of biliary and duodenal obstruction. In describing the utility of a staging modality such as laparoscopy in the context of the AJCC system, it is therefore of primary importance to consider the degree to which it can detect mesenteric arterial invasion and the presence of distant metastases, as both of these findings are associated with an upstaging of disease and a consequent change in the strategies available for treatment. When implemented appropriately, a comprehensive staging algorithm incorporating both noninvasive imaging and selective exploratory laparoscopy can be highly effective in this regard.

3

Techniques of Laparoscopic Staging

The laparoscopic techniques used for staging pancreatic malignancy have evolved over the past two decades. The procedure may be an outpatient study performed several days prior to potential open resection or it can be performed under the same

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general anesthetic as the open operation. Simple and extended techniques have been described (15, 16). We prefer a simple exploratory procedure performed under the same general anesthetic as potential resection. Pneumoperitoneum is initially obtained via an open Hasson technique or by using a Veress needle. Peritoneal access is typically through an infraumbilical incision, although a history of previous abdominal operations may necessitate a change in strategy. Once a single trocar has been placed, meticulous inspection of the liver, greater omentum, diaphragm, and abdominal and pelvic peritoneal surfaces is performed. A second 5-mm trochar, typically placed through a right upper quadrant incision, may be placed to manipulate a retracting device. Suspicious nodules are biopsied using a percutaneously inserted Tru-Cut needle. We do not typically perform an extensive dissection or laparoscopic ultrasonography, nor do we routinely obtain peritoneal aspirates for cytologic examination. Patients are considered to have unresectable disease in the presence of peritoneal or liver metastases (Fig. 16.1); those patients in whom these are not identified undergo immediate open exploration with the hope of curative resection. The entire procedure takes approximately 10–15 minutes of operating time. Extended procedures, such as that advocated by the group at Memorial SloanKettering Cancer Center, are grounded on the belief that the technique of laparoscopic staging should mimic the operative assessment performed at exploratory laparotomy (15, 17). A four-trocar setup is typically used. Prior to tumor manipulation, peritoneal washings are obtained for cytologic assessment. The primary tumor is then completely assessed, as are all peritoneal surfaces and the surface of the liver. Inspection of the mesocolon and middle colic vein is followed by division of the gastrohepatic ligament to expose the lesser sac, caudate lobe of the liver, vena cava, and celiac axis. Finally, laparoscopic ultrasound is performed to evaluate the liver and pancreas, with particular attention paid to the relationship of the primary tumor to major vessels. Biopsy of any suspicious nodes or lesions is performed at any point in the procedure. Patients are considered to have unresectable disease if

a

b

Fig. 16.1 A Surface liver lesion (arrow) discovered on laparoscopy in a 73-year-old man who was found on CT scan to have a potentially resectable mass in the head of the pancreas as well as several ambiguous, subcentimeter cystic lesions in the liver. B Frozen section biopsy of this liver lesion confirmed metastatic pancreatic cancer and the patient underwent endoscopic palliation of his jaundice

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they have histologically confirmed metastases, extrapancreatic extension of tumor, celiac or high portal node involvement, or invasion or encasement of the major visceral vessels. Although it offers the surgeon the ability to more thoroughly evaluate the tumor, this technique takes considerably longer, and the dissection that it requires demands advanced laparoscopic skills.

4 Efficacy and Limitations of Noninvasive Staging Because staging laparoscopy is an adjunctive staging study, and as such is not intended to replace currently available noninvasive techniques, it is important to consider the strengths and limitations of these existing staging modalities. Today, the diagnostic algorithm for staging pancreatic cancer is based upon abdominal imaging using a high-resolution, multidetector CT scanner. Modern protocols employ helical scanners, with which dual phase cross-sectional imaging of the pancreas and liver in the arterial and portal venous phases can be performed to detect both primary and metastatic disease with high sensitivity. In an evaluation of recent studies assessing helical CT in the diagnosis of pancreatic cancer, Tamm (18) found the overall detection rate to be approximately 89% and 71% for cancers >2 cm and 80 years old to nearly 2,500 patients 8.1 kg in the previous 6 months and serum albumin levels of 5.2 pg/ml had a significantly worse survival compared with patients with lower levels. Higher levels were also associated with a poor performance status and/or weight loss (28). IL-6 together with IL-8 are important proinflammatory cytokines involved in the induction of the APR. IL-6 has the potential to act as a cachectic factor. Atrophy of muscle is observed in IL-6 transgenic mice, which is completely blocked by anti-mouse IL-6 receptor antibody (37). Muscle atrophy was associated with increased mRNA levels for cathepsins B and L, as well as mRNA levels of poly and monoubiquitin, although other studies (32), in which IL-6 was administered IV into rats could not detect any changes in expression of ubiquitin mRNA. An increase in whole body lipolysis and/or fat oxidation was observed after infusion of recombinant human IL-6 in resting humans (38). As with TNF-α this effect could result from inhibition of LPL (33), although IL-6 may also directly increase lipolysis and fat oxidation in adipocytes. Evidence for a role for IL-6 in cancer cachexia has mainly come from the murine colon-26 adenocarcinoma model, although such studies suggest that IL-6 may not act alone to produce cachexia, and that an additional unknown factor may be responsible (39).

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Other Cytokines

A number of other cytokines including interleukin-1 (IL-1) and interferon-γ (IFNγ) have been implicated in the induction of cachexia in animal models. However, as with TNF-α no correlation has been found between serum levels of IL-1 or IFN-γ and cachexia or anorexia in patients with advanced and terminal cancer (40), although both cytokines induced ubiquitin mRNA expression in the skeletal muscle of rats, and thus have the potential to induce protein degradation (32). The serum concentration of soluble IL-2 receptor has been found to be highest in cachectic patients and was inversely correlated with the serum concentration of nutritional parameters such as prealbumin and transferrin (41). The length of survival of cachectic patients was inversely correlated with the serum concentration of soluble IL-2 receptor.

5.4

Zinc a 2-Glycoprotein

The search for a lipid mobilizing factor (LMF), associated with cancer cachexia, led to the isolation of a 43-kDa glycoprotein from the urine of cancer patients with weight loss, which was subsequently shown to be zinc α2-glycoprotein (ZAG) (42). LMF/ZAG stimulates lipolysis directly in isolated adipocytes, through stimulation of adenylate cyclase, in a GTP-dependent process, through interaction with the β3adrenergic receptor (β3-AR) (43). ZAG also induces an increase in expression of UCP-1 and -2 in isolated BAT, through interaction with the β3-AR, and an increase in UCP-3 in murine myotubes through activation of mitogen activated protein kinase (44). In vivo studies using obese male mice showed ZAG to induce a loss of body weight, without a reduction in food intake, which was attributed entirely to the loss of body fat, without a reduction in food and water intake (45). This was attributed to the lipolytic effect of ZAG coupled with an increase in energy expenditure, due to induction of UCP-1 expression in BAT. ZAG is expressed not only by cachexiainducing tumors, but also by white adipose tissue (WAT) and BAT (46). Moreover, ZAG expression in WAT and BAT increased 10- and 3-fold, respectively, in cachectic mice, which had lost 19% of their body weight, and 60% of their adipose tissue. This increase in ZAG expression appears to be induced by glucocorticoids (47), and the glucocorticoid receptor antagonist, RU38486, attenuated both the loss of body weight and increase in ZAG expression in WAT in mice bearing a cachexia-inducing tumour. Other anti-cachectic agents such as eicosapentaenoic acid (EPA) also downregulate ZAG expression in WAT and BAT by opposing the action of glucocorticoids (48). There have been no direct measurements of circulating ZAG levels in cachectic cancer patients, although the ability of serum or urine to induce lipolysis in isolated adipocytes was found to be significantly higher (twofold) in cancer patients with weight loss, compared with weight-losing patients with Alzheimer’s disease, non– weight-losing cancer patients or healthy controls (49).

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5.5

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Proteolysis-Inducing Factor

Bioassays of serum from cancer patients with a weight loss >10% provided evidence for a circulating factor capable of producing protein degradation in isolated gastrocnemius muscle (50). This factor, named proteolysis-inducing factor (PIF), was subsequently isolated from the urine of cachectic patients with pancreatic cancer, and was shown to be a sulfated glycoprotein of molecular mass 24 kDa, and unlike any of the known cytokines (51). Although PIF has been identified in the urine of weight-losing patients with other types of cancer, it was not detectable in other weight-losing conditions such as sepsis, multiple injuries, surgery, or sleeping sickness (52). Experiments in mice suggest that PIF may be an embryonic protein expressed during the stage of patterning and eventual development of skeletal muscle, and that it may be abnormally expressed in certain tumors (53). Administration of PIF to normal mice caused profound weight loss (about 10% in 24 hours) without a reduction in food and water intake. The major contributor to the loss of body weight was lean body mass, and this was found to arise from a depression in protein synthesis (by 50%) and an increase in protein degradation (by 50%), as occurs in the skeletal muscle of cachectic cancer patients (54). Unlike the cytokines PIF induces protein degradation directly in isolated murine myotubes, and the effect both in vitro and in vivo is due to an increase in activity and expression of the ubiquitin-proteasome proteolytic pathway (55). PIF induces an intracellular signalling pathway in skeletal muscle, which results in the activation of the transcription factor nuclear factor-κB (NF-κB), leading to increased expression of proteasome subunits and ubiquitin conjugating enzyme (56). PIF has been detected in the urine of 80% of patients with pancreatic cancer, and these patients had a significantly greater total weight loss and rate of weight loss than patients whose urine did not contain PIF, but this was not associated with a reduced survival (57). PIF also activates NF-κB and the transcription factor STAT3 in human hepatocytes, resulting in the increased production of IL-6, IL-8, and CRP, and the decreased production of transferrin (53). Thus PIF is likely to be involved in the proinflammatory response in cachexia.

5.6

Angiotensin II

Angiotensin II (Ang II) is capable of causing anorexia and wasting in animal models with a decrease in lean body mass arising from an increased protein breakdown (58). Protein degradation is mediated through the ubiquitin-proteasome proteolytic pathway, since mRNA levels of the ubiquitin-ligases atrogin-1 and muscle ringfinger-1 (MuRF1) were upregulated in skeletal muscle by Ang II (59). Ang II also directly induces protein degradation in vitro, suggesting a direct effect, and the cellular signaling pathway leading to increased proteasome expression and activity appears to be the same as with PIF, through activation of NF-κB (60). Ang II has also been shown to directly inhibit protein synthesis in murine myotubes and the

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effect is attenuated by insulin-like growth factor 1 (IGF-1) (61). There are no reports of Ang II having an effect on adipose tissue.

6 Agents Used in the Treatment of Cachexia 6.1

Progestins

The progestational agents megestrol acetate (Megace) and medroxyprogesterone acetate (MPA) are appetite stimulants and have been evaluated in the treatment of cancer cachexia/anorexia. All studies with Megace report an increase in appetite and weight gain of variable degrees (62). However, some studies with MPA, although reporting an improvement in appetite, did not find any weight gain (63). Long-term treatment with Megace (>12 weeks) results in side effects, principally hypertension and edema resulting from water retention, as well as thromboembolic events (62). For both Megace (64) and MPA (65) measurements of body composition using dual energy X-ray absorptiometry and tritiated body water at the time of weight gain showed that the majority of the gained weight resulted from an increase in adipose tissue, and some body fluid, but there was no increase in lean body mass. This explains why the performance status (Karnofsky index) was not improved with either agent. MPA has been shown to reduce levels of the cytokines IL-1β, IL6, and TNF-α produced by peripheral blood mononuclear cells of cancer patients in response to phytohemagglutinin stimulation (66). This result may explain the appetite promoting effect of progestational agents.

6.2

Eicosapentaenoic Acid

Animal studies have shown that of the two principal omega-3 fatty acids found in fish oil, only eicosapentaenoic acid (EPA) has an anticachectic effect, whereas DHA is devoid of therapeutic activity in this area (67). Despite this the two omega3 fatty acids are often used in combination in clinical studies and sometimes the dose is calculated as the total omega-3 fatty acids rather than EPA alone. However, there have been promising results in the treatment of cachexia with fish oil, particularly for patients with pancreatic cancer. The optimum dose of EPA is around 2 g per day, or slightly higher, and treatment needs to proceed for at least 3 weeks for a therapeutic benefit to be seen. Treatment with EPA alone stabilizes body weight, whereas the combination of EPA with a caloric and protein enriched nutritional supplement produces an increase in body weight of up to 2 kg after 7 weeks of treatment (68). Unlike Megace, the increase in body weight was attributed to an increase in lean body mass, with no change in fat mass, and also unlike Megace there was a statistically significant improvement in Karnofsky performance status.

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Patients taking the EPA supplement that gained weight reported an improved quality of life, and this was reflected by an increase in TEE and PAL (11). There was a considerable improvement in the survival of both cachectic and non-cachectic subjects with generalized solid tumors receiving a daily supplement of 18 g omega-3 fatty acids (69), and treatment also increased the ratio of T-helper cells to T-suppressor cells in cachectic subjects. Although EPA is thought to work by attenuating the action of PIF (70), which does not induce anorexia, clinical studies have shown EPA to be as effective as Megace in improving appetite (71).

6.3

Thalidomide

Thalidomide has been shown to be effective in attenuating loss of body weight in patients with advanced pancreatic cancer who had lost at least 10% of their body weight (72). As with EPA thalidomide produced weight stabilization rather than producing large weight gain and anthropometric measurements suggested that the effect was on lean body mass. Thalidomide has also been shown to improve lean body mass in a small uncontrolled study in patients with nonobstructing and inoperable esophageal cancer (73). Thalidomide has the potential to reduce production of TNF-α by increasing the degradation rate of TNF mRNA, but it also blocks NF-κB regulated genes through suppression of IκB kinase activity (74). An effect on TNFα production seems unlikely since pentoxifylline, a methylxanthine derivative, also shown to inhibit TNF-α production, had no effect on appetite or body weight of cachectic cancer patients (75). The most likely mechanism for the effect of thalidomide is due to suppression of protein degradation induced by factors such as PIF (56) and Ang II (60) by attenuating activation of NF-κB and the subsequent increase in proteasome and MuRF1 expression.

6.4

b-Hydroxy-b-Methylbutyrate

β-Hydroxy-β-methylbutyrate (HMB) is formed by transamination of leucine to αketoisocaproate in muscle, followed by oxidation of the α-ketoisocaproate in the liver cytosol to HMB. HMB, combined with arginine and glutamine, has been shown to be effective in increasing body weight through an increase in lean body mass in weight-losing patients with advanced (stage IV) cancer (76). No other clinical studies have been reported in cancer cachexia, although HMB has also been shown to attenuate AIDS-associated wasting. In an experimental model of cancer cachexia HMB has been shown to attenuate both muscle protein degradation and the depression in protein synthesis (77). The effect on protein degradation was due to attenuation of the up-regulation of the ubiquitin-proteasome pathway. HMB appears to act like EPA to inhibit cellular signalling induced by PIF leading to activation of NF-κB (78).

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Appetite Stimulants

Dronabinol, the active ingredient in marijuana, at a dose of 5 mg daily caused a subjective improvement in both mood and appetite in patients with unresectable cancer, but all patients showed a progressive loss of body weight (79). In a placebocontrolled trial, the serotonin antagonist cyproheptadine, although having a small effect on appetite, failed to significantly abate progressive weight loss in cancer patients (80). Ghrelin is a brain-gut peptide that stimulates appetite by activating neurons of the arcuate nucleus of the hypothalamus, an area that regulates feeding. When administered to patients with cancer anorexia it increases energy intake by about 30% without any side effects (81). Although there were no reported effects on body weight, in nude mice bearing the SEKI human melanoma, ghrelin suppressed weight loss through an increase in fat mass with no reported increase in muscle mass (82). These results are similar to those reported with the appetite stimulants Megace and MPA. None of the other orexigenic neuropeptides has yet undergone clinical evaluation.

7 Conclusion Despite the introduction of new treatments cachexia and anorexia still remain formidable clinical problems in the management of cancer patients. Knowledge of the mechanism of this syndrome is important in the future clinical development of new forms of treatment. Unfortunately in the quest to treat the cancer, cachexia and anorexia are often overlooked as being an inevitable consequence of the disease. If treatment is successful, then the syndrome disappears, but at the present time for patients with pancreatic cancer this rarely happens, and anti-cachexia therapy should be a part of the armaments for the treatment of this disease.

References 1. Wys WD, De Begg C, Lowin PT, 1980, Prognostic effect of weight loss prior to chemotherapy in cancer patients. Am J Med 69:491–497. 2. Wigmore SJ, Plester CE, Richardson RA, 1997, Changes in nutritional status associated with unresectable pancreatic cancer. Br J Cancer 75:106–109. 3. Vigano A, Bruera E, Jhangri GS, 2000, Clinical survival predictors in patients with advanced cancer. Arch Int Med 160:861–868. 4. Barber MD, Ross JA, Fearon KCH, 1999, Changes in nutritional, functional and inflammatory markers in advanced pancreatic cancer. Nutr Cancer 35:106–110. 5. Persson C, Glimelius B, 2002, The relevance of weight loss for survival and quality of life in patients with advanced gastrointestinal cancer treated with palliative chemotherapy. Anticancer Res 22:3661–3668. 6. Okusaka T, Okada S, Ishii H, 1998, Prognosis of advanced pancreatic cancer patients with reference to calorie intake. Nutr Cancer 32:55–58.

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31. Busquets S, Sanchis D, Alvarez B, 1998, In the rat, tumor necrosis factor α administration results in an increase in both UCP-2 and UCP-3 mRNA in skeletal muscle: a possible mechanism for cytokine-induced thermogenesis? FEBS Lett 440:348–350. 32. Llovera M, Carbo N, Lopez-Sorino J, 1998, Different cytokines modulate ubiquitin gene expression in rat skeletal muscle. Cancer Lett 13:83–87. 33. Berg M, Fraker DL, Alexander HR, 1994, Characterisation of differentiation factor/leukaemia inhibitory factor effect on lipoprotein lipase activity and mRNA in 3T3-L1 adipocytes. Cytokine 6:425–432. 34. Zhang HH, Halbleib M, Ahmad R, 2002, Tumor necrosis factor-α stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cyclic AMP. Diabetes 51:2929–2935. 35. Scott HR, McMillan DC, Crilly A, 1996, The relationship between weight loss and interleukin 6 in non-small-cell lung cancer. Br J Cancer 73:1560–1562. 36. Iwase S, Murakami T, Sato Y, 2004, Steep elevation of blood interleukin-6 (IL-6) associated with late stages of cachexia in cancer patients. Eur Cytokine Netw 15:312–316. 37. Tsujinaka T, Fujita J, Ebisui C, 1996, Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice. J Clin Invest 97:244–249. 38. Hall G, Van Steensberg A, Sacchetti M, 2003, Interleukin-6 stimulates lipolysis and fat oxidation in humans. J Clin Endocrinol Metab 88:3005–3010. 39. Fujiki F, Mukaida N, Hirose K, 1997, Prevention of adenocarcinoma colon 26-induced cachexia by interleukin 10 gene transfer. Cancer Res 57:94–99. 40. Maltoni M, Fabbri L, Nanni O, 1997, Serum levels of tumour necrosis factor alpha and other cytokines do not correlate with weight loss and anorexia in cancer patients. Support Care Cancer 5:130–135. 41. Shibata M, Takekawa M. 1999, Increased serum concentration of circulating soluble receptor for interleukin-2 and its effect as a prognostic indicator in cachectic patients with gastric and colorectal cancer. Oncology 56:54–58. 42. Todorov PT, McDevitt TM, Meyer DJ, 1998, Purification and characterisation of a tumor-lipid mobilising factor. Cancer Res 58:2353–2358. 43. Russell ST, Hirai K, Tisdale MJ, 2002, Role of β3-adrenergic receptors in the action of a tumour lipid mobilising factor. Br J Cancer 86:424–428. 44. Sanders PM, Tisdale MJ, 2004, Effect of zinc-α2-glycoprotein (ZAG) on expression of uncoupling proteins in skeletal muscle and adipose tissue. Cancer Lett 212:71–81. 45. Russell ST, Zimmerman TP, Domin BA, 2004, Induction of lipolysis in vitro and loss of body fat in vivo by zinc-α2-glycoprotein. Biochim Biophys Acta 1636:59–68. 46. Bing C, Jenkins J, Sanders P, 2004, Zinc-α2-glycoprotein, a lipid mobilising factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia. Proc Natl Acad Sci U S A 101:2500–2505. 47. Russell ST, Tisdale MJ, 2005, The role of glucocorticoids in the induction of zinc-α2-glycoprotein expression in adipose tissue in cancer cachexia. Br J Cancer 92:876–881. 48. Russell ST, Tisdale MJ, 2005, Effect of eicosapentaenoic acid (EPA) on expression of a lipid mobilising factor in adipose tissue in cancer cachexia. Prostaglandins, Leukotrines, Essential Fatty Acids 72:409–414. 49. Groundwater P, Beck SA, Barton C, 1990, Alteration of serum and urinary lipolytic activity with weight loss in cachectic cancer patients. Br J Cancer 62:816–821. 50. Belizario JE, Katz M, Raw CI, 1991, Bioactivity of skeletal muscle proteolysis-inducing factors in the plasma proteins from cancer patients with weight loss. Br J Cancer 63:705–709. 51. Todorov P, Caruik P, McDevitt T, 1996, Characterisation of a cancer cachectic factor. Nature 379:739–742. 52. Caruik P, Lorite MJ, Todorov PT, 1997, Induction of cachexia in mice by a product isolated from the urine of cachectic cancer patients. Br J Cancer 76:606–613. 53. Watchorn TM, Waddell ID, Dowidar N, 2001, Proteolysis-inducing factor regulates hepatic gene expression via the transcription factors NF-κB and STAT3. FASEB J 15:562–564.

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54. Lorite MJ, Caruik P, Tisdale MJ, 1997, Induction of muscle protein degradation by a tumour factor. Br J Cancer 76:1035–1040. 55. Lorite MJ, Smith HJ, Arnold JA, 2001, Activation of ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor (PIF). Br J Cancer 85:297–302. 56. Wyke SM, Tisdale MJ, 2005, NF-kB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle. Br J Cancer 92:711–721. 57. Wigmore SJ, Todorov PT, Barber MD, 2000, Characteristics of patients with pancreatic cancer expressing a novel cancer cachectic factor. Br J Surg 87:53–58. 58. Brink M, Price SR, Chrast J, 2001, Angiotensin II induces skeletal muscle wasting through enhanced protein degradation and down-regulates autocrine insulin-like growth factor 1. Endocrinology 142:1489–1496. 59. Song Y-H, Li Y, Du J, 2005, Muscle-specific expression of IGF-1 blocks angiotensin-IIinduced skeletal muscle wasting. J Clin Invest 115:451–458. 60. Russell ST, Wyke SM, Tisdale MJ, 2006, Mechanism of induction of muscle protein degradation by angiotensin II. Cell Sig 18:1087–1096. 61. Russell ST, Sanders PM, Tisdale MJ, 2006, Angiotensin II directly inhibits protein synthesis in murine myotubes. Cancer Lett 231:290–294. 62. Maltoni M, Nanni O, Scarpi E, 2001, High-dose progestins for the treatment of cancer anorexia-cachexia syndrome: A systematic review of randomised clinical trials. Ann Oncol 12:289–300. 63. Downer S, Joel S, Albright A, 1993, A double blind placebo controlled trial of medroxyprogesterone acetate (MPA) in cancer cachexia. Br J Cancer 67:1102–1105. 64. Loprinzi CL, Schaid DJ, Dose AM, 1993, Body composition changes in patients who gain weight while receiving megestrol acetate. J Clin Oncol 11:152–154. 65. Simms JPFHA, Schols AMJ, Hoefangels JMJ, 1998, Effects of medroxyprogesterone acetate on food intake, body composition and resting energy-expenditure in patients with advanced nonhormone-sensitive cancer. Cancer 82:553–560. 66. Montovani G, Maccio A, Esu S, 1997, Medroxyprogesterone acetate reduces in vitro production of cytokines and serotonin involved in anorexia/cachexia and emesis by peripheral blood mononuclear cells of cancer patients. Eur J Cancer 33:602–607. 67. Beck SA, Smith KL, Tisdale MJ, 1991, Anticachetic and antitumour effect of eicosapentaenoic acid and its effect on protein turnover. Cancer Res 51:6089–6093. 68. Barber MD, Ross JA, Voss AC, 1999, The effect of an oral nutritional supplement enriched with fish oil on weight-loss in patients with pancreatic cancer. Br J Cancer 81:80–86. 69. Gogos CA, Ginopoulos P, Salsa B, 1998, Dietary omega-3 polyunsaturated fatty acids plus vitamin E restore immunodeficiency and prolong survival for severely ill patients with generalised malignancy. Cancer 82:395–402. 70. Smith HJ, Lorite MJ, Tisdale MJ. 1999, Effect of a cancer cachectic factor on protein synthesis/degradation in murine C2. C12 myoblasts: Modulation by eicosapentaenoic acid Cancer Res 59:5507–5513. 71. Jatoi A, Rowland K, Loprinzi Cl, 2004, An eicosapentaenoic acid supplement versus megestrol acetate versus both for patients with cancer-associated wasting: a North Central Cancer Treatment Group and National Cancer Institute of Canada collaborative effort. J Clin Oncol 22:2469–2476. 72. Gordon JN, Trebble TM, Ellis RD, 2005, Thalidomide in the treatment of cancer cachexia: A randomised placebo controlled trial. Gut 54:540–545. 73. Khan ZH, Simpson EJ, Cole AT, 2003, Oesophageal cancer and cachexia. The effect of shortterm treatment with thalidomide on weight loss and lean body mass. Aliment Pharmacol Ther 17:677–682. 74. Keifer JA, Guttridge DC, Ashburner BP, 2001, Inhibition of NF-kB kinase activity. J Biol Chem 276:22382–22387.

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Chapter 27

Endoscopic Palliation Nathan Schmulewitz

1 Introduction Pancreatic duct adenocarcinoma will be diagnosed in nearly 34,000 persons in the United States this year, and almost the same number will die of their disease (1). Pancreatic cancer has an overall survival rate of < 4% (2), and even in patients with tumors truly limited to the pancreas and without nodal disease, actuarial 5-year survival rates are 18–24% (3). In fact, nearly 85% of patients are deemed unsuitable for potentially curative resection at time of diagnosis (4–6). Therefore, palliation of symptoms and/or imminent morbidity is the cornerstone of care for most patients with pancreatic cancer. Furthermore, the absence of therapeutic interventions is a predictor of decreased survival (7). With these facts as a backdrop, this chapter discusses the role of endoscopic interventions in the management of advanced pancreatic malignancy.

2 Management of Biliary Obstruction Between 70% and 90% of patients with pancreatic adenocarcinoma present with biliary obstruction (8). Left alone, biliary obstruction leads to jaundice, pruritus, cholangitis, liver dysfunction, anorexia, malabsorption, and altered coagulation. These symptoms tend to have a profound effect on quality of life, and so biliary drainage procedures are generally appropriate for patients who are not candidates for resection (9, 10). Biliary drainage can be accomplished in two ways, surgical bypass with biliary-enteric anastomosis or placement of an endoprosthesis. Endoprosthetic drainage of the bile duct can be accomplished by percutaneous or transduodenal approaches. Overall, the most durable therapy for malignant biliary obstruction is surgical bypass. Some studies have shown recurrent biliary obstruction rates as low as 2% (11, 12). However, perioperative morbidity and mortality rates run from 20% to 40% and 10% to 24%, respectively (11, 13–19). Other concerns with surgical therapy have included prolonged ileus, prolonged hospitalizations, and expense. One

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recent surgical series only included patients with pancreatic head cancers who were taken to the operating room for potentially curative surgery and examined outcomes of 83 consecutive patients who were found unresectable and underwent both biliary-enteric bypass and gastrojejunostomy (12). In that series, 30-day mortality was 4.8%, perioperative morbidity 27%, delayed gastric emptying occurred in 9%, and the mean hospital stay was 16 days. To try to minimize the morbidity of surgery, there has been some experience with laparoscopic biliary bypass, but this has not been widely used as the morbidity still far surpasses that of nonoperative biliary decompression (20). For nonoperative biliary decompression, percutaneous, transhepatic placement of drainage tubes and stents is an alternative with a high success rate. Relief of biliary obstruction can be achieved in >97% of patients with this approach (21–24). Self-expanding metallic stents are favored, as they obviate the need for permanent external catheters, thereby decreasing complications of leakage, infection, and hemorrhage, and eliminate concerns of tube dislodgement, migration, or blockage, in addition to improving the patient’s psychological condition (25) Metallic-stent mean patency rates range from 129 to 324 days (22, 24, 26). Nevertheless, although metallic stents reduce morbidity and mortality over plastic stents (26), presumedly due to improved patency, 30-day mortality rates range from 7.5% to 36% (24, 26, 27), and complication rates can be significantly higher. There are several other shortcomings to the percutaneous transhepatic approach. First, although biliary access, drainage, and stent placement are usually possible, transpapillary stent placement is not always possible, condemning some patients to lifelong external drainage. Second, even when transpapillary stent placement is successful, the patient usually has to return for several procedural sessions, including dilations prior to metallic stent placement and then for removal of external catheter(s). The temporary presence of external drains can cause significant problems, such as dislodgements, infections, leaking, or bleeding. More recently, efforts have been made to study the feasibility of placing a stent at the initial percutaneous procedure, although a temporary percutaneous catheter is generally still required for hemostatic purposes (28). Nevertheless, the percutaneous approach for biliary drainage remains an important therapeutic option and is particularly important for several patient subsets. These special patient groups include those with acute cholangitis and no immediate access to endoscopic decompression, those failing endoscopic decompression (29), and those with postsurgical anatomy limiting enteric access to the bile duct. For most unresectable pancreatic cancer patients with biliary obstruction, endoscopic decompression is the procedure of choice. The bile duct is accessed via the major papilla in the duodenum using endoscopic retrograde cholangiography (ERC). During the procedure, a catheter and/or wire is passed across the papilla and into the biliary tree and across the malignant stricture, under fluoroscopy. Thereafter, a stent can be guided over the wire to bridge the stricture, allowing the distal end of the stent to hang into the duodenum. Traditionally, biliary stents have been made of plastic. Plastic stents can be placed with a >93% technical success rate, and do not require biliary sphincterotomy (30). When endoscopic access into

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the bile duct or across the stricture is unsuccessful, temporary percutaneous drainage with passage of a tube or wire across the stricture and into the duodenum frequently allows for subsequent endoscopic stenting. Once endoscopic stent placement is achieved, any external drain can be removed. Unfortunately, plastic stents have high occlusion rates resulting from development of a biofilm on the internal surface leading to bacterial colonization (31, 32). Bacteria lead to calcium bilirubinate precipitation with sludge accumulation. Bilioduodenal reflux and food may also play a role in stent occlusion (33, 34). Because of such inevitable occlusions, standard 10 French (F) plastic stents generally stay patent for 3–4 months (35, 36). Furthermore, changes in plastic stent composition, surface features, and even use of antibiotics have not significantly affected patency (37–39). As a result, obstructive jaundice recurs in up to 50% of patients, requiring that plastic stents be changed every several months (40). With the evolution of self-expandable metal stents (SEMS), there has been significant improvement in stent patency. SEMS are made of stainless steel alloy or nickel-titanium alloy. They come packaged within a 7.5- to 10-F sheath and are passed over a wire into the bile duct and across the stricture, after biliary sphincterotomy. Thereafter, the sheath is retracted and the stent expands as it is deployed, up to 10 mm in diameter. As the metal struts press into surrounding tissue, and granulation tissue and tumor grows between the struts, the stent becomes fixed to its surroundings and thereby is usually considered permanent. Having a larger internal bore, these stents can maintain patency for an average of 9 months (41–44). Like the plastic stents, SEMS can be placed with a very high technical success rate. Unfortunately, SEMS are far more expensive than plastic stents. Cost-effectiveness studies have shown that metal stents are only economical and appropriate when the patients are expected to survive 6 months or longer (45–47). Some also advise routine placement of a SEMS after occlusion of a plastic stent. Two other appropriate situations for metal stents include management of a patient who might not comply with medical follow-up and the patient with impending duodenal obstruction (35). The latter scenario is an important one, because endoscopic placement of a biliary stent in a patient with duodenal obstruction or after duodenal enteral stent placement can be exceedingly difficult. Of course, even metal stents succumb to occlusion via tumor ingrowth, overgrowth at the ends, mucosal hyperplasia, biliary sludge, or food impaction (35). When a SEMS becomes occluded, mechanical “cleaning” can be performed endoscopically, although that process alone is rarely efficacious. More frequently, stent cleaning is followed by placing another metal stent within the first one or by placing a plastic stent within the metal stent (48). Although both of these stent-in-astent techniques are successful >90% of the time, deploying a plastic stent within the SEMS tends to be more cost effective (48, 49). Polyethylene and silicone covers for SEMS have been tried to reduce tissue ingrowth through the wire mesh, generally without significant improvements in patency (50–52). One study of covered versus uncovered nickel-titanium SEMS did show a patency advantage to the covered stents, but the covered stents also led to more complications (53). There are three downsides to covered metals stents. First,

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covered SEMS are more prone to migration, as a result of poor friction between the polymer surface and the tumor. Second, covered SEMS can predispose to acute cholecystitis or pancreatitis, with incidences as high as 4.2% and 8.7%, respectively, presumably when a portion of the covered section blocks the cystic duct or pancreatic duct orifice (53, 54). Third, unlike wire mesh stents that “dig” into adjacent tissue, facilitating full expansion, covered stents, even when only the internal surface is covered, cannot cut deeply into adjacent tissue, which may occasionally limit full radial deployment (55). Obviously a stent with a narrower lumen will be more prone to occlusion due to biofilm and sludge. Luckily, coated SEMS are usually retrievable, although it can certainly be difficult to place these stents so that the coated portion does not cover the cystic duct or ampulla. Unfortunately, endoscopic stenting of biliary obstruction in pancreatic cancer is certainly not without complications. Overall complication rates range from 15% to 34% (56), and the range of adverse events is broad. Procedurally related complications can include cardiopulmonary compromise, hemorrhage, cholangitis, pancreatitis, and perforation. Stent placement in malignant biliary obstruction is a significant risk factor for cholangitis (57), particularly if complete drainage cannot be achieved, and all patients should receive periprocedural broad-spectrum antibiotics, if they are not already on antibiotics for their biliary obstruction. Additionally, endoscopic sphincterotomy is needed for metallic stent placement, and sphincterotomy is a risk factor for hemorrhage and perforation (57). Indeed this patient group has the highest 30-day mortality rates of ERC cohorts. Beyond intraprocedural complications, untoward outcomes in this patient group can include cholangitis, stent migration, recurrent obstruction, and frank failure to adequately drain the bile duct. Although cholangitis is usually of greatest concern prior to therapy for biliary obstruction, it can also occur in patients after therapeutic stenting, even in the context of stent patency. Patients at highest risk are those with a SEMS across the major papilla (58). The mechanism might be related to duodenal contents, food, or digestive juices traveling into the biliary tree. Of course, adequate biliary drainage is the most common short- and long-term concern in biliary obstruction. Although some studies demonstrate ERC to have therapeutic success for stent placement in well over 90% of cases, other studies have shown failure rates as high as 13% or more (29). Percutaneous drainage and attempted stenting is generally the preferred approach in these patients not technically amenable to successful endoscopic drainage. One other approach to failed biliary cannulation by ERC is biliary access gained through use of endoscopic ultrasound (EUS). The roles of EUS in periampullary neoplasms has traditionally been for staging and tissue acquisition via fine-needle aspiration (FNA). However, with its ability to image the biliary tree in real time, EUS can also be used to gain access into the biliary tree. A FNA needle or needle knife can be passed through the wall of the duodenum directly into the bile duct, or through the wall of the stomach into a distended left intrahepatic duct (59–62). Once access is achieved with a hollow-bore needle, a wire can be passed into the duct, under fluoroscopy. Sometimes the wire can be guided through the stricture and into the duodenum, where it can be captured by a traditional duodenoscope and

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used to guide transpapillary stenting via ERC (i.e., a “rendezvous” procedure). When transpapillary passage of the wire is not possible, the newly formed cholangioenteric fistula can be dilated over the wire and a drainage catheter or stent can be placed for biliary drainage. The allure of EUS-guided biliary drainage after failed ERC-guided stenting is the avoidance of percutaneous catheter drainage and its associated morbidity. However, the role for this technique in the salvage management of malignant biliary obstruction is yet unclear. In summary, endoscopic biliary stenting via ERC is the preferred route for palliative management of biliary obstruction in patients with unresectable pancreatic cancer. Despite a higher incidence or recurrent obstruction, endoscopic stenting affords lower morbidity, shorter hospitalization and lower 30-day mortality than surgical bypass with similar technical success and quality of life (47). Endoscopic stenting also has fewer complications than percutaneous stenting (27), in addition to requiring fewer sessions and not obligating patients to have temporary external catheters. Plastic stents tend to remain patent for 3–4 months, whereas the more costly metal stents can be expected to remain patent for up to 9 months, and the latter usually do not require retreatment thereafter (63). Occlusions to either plastic or metal stents can be managed endoscopically. Patients expected to survive >6 months would likely benefit most from SEMS, although some other groups are also appropriate candidates for having metallic stents placed initially. Some covered metal stents might further delay recurrent obstruction, but there are not enough data to recommend their use at this time. Biliary obstruction with failed attempts at ERC stenting is usually best managed percutaneously, although endoscopic and surgical options are also available.

3 Management of Gastric Outlet Obstruction Traditionally, gastroduodenal obstruction in cases of unresectable malignancy was managed with open surgical gastrojejunostomy. Placement of a surgically deployed plastic prosthesis for palliation of obstructive gastric cancer was first described in 1980 (64). Eventually, use of endoscopically placed self-expanding metallic stents for malignant gastroduodenal obstruction became popularized in the early 1990s. Since then, there have been many published series of patients managed with endoscopic stenting. The procedure can be done in two ways, depending on patient anatomy and available devices. One technique is to pass an endoscope to the level of obstruction and then pass a wire across the stenosis, under fluoroscopy. Typically, the distal or proximal aspect of the stenosis is marked with a radiopaque marker either internally, as with a metallic hemoclip, or externally, as can be done with a paper clip taped to the patient so as to lie over the stricture during fluoroscopy. After the scope is backed out of the patient over the wire, a sheathed SEMS is carefully passed over the wire, using fluoroscopic guidance to deploy the stent across the stenosis. The second technique is to use a through-the-scope SEMS device that can be pushed out of the endoscope channel and visually guided, with or without fluoroscopy, to the appropriate site of deployment.

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The largest single series of endoscopically placed SEMS for malignant gastroduodenal obstruction was a retrospective study of 176 patients treated over 7 years at four centers (65). Technical success was achieved in 98% of patients, and procedure-related complications occurred in 14 patients. These complications included stent migration, bowel perforation, intestinal bleeding, and abdominal pain. Another nine patients (6%) developed cholangitis during the follow-up period. Regarding the patients for whom follow-up data was available, 84% resumed oral intake for a median time of 146 days, 22% of patients were restented, and 60% of the patients who died during follow-up were tolerating oral intake up until their deaths. Importantly, the only factor significantly predictive of prolonged oral intake was chemotherapy after stent placement. Another series of 63 patients with malignant gastric outlet obstruction treated endoscopically showed similar results (66). In this study there was 95% technical success, with 92% of patients taking exclusively peroral nutrition and 73% tolerating soft or solid food. Complications occurred in 30% of patients, including two perforations, 13 stent obstructions, and four stent migrations. One systematic review looked at 32 published case series of gastroduodenal obstruction managed with metallic stents (67). Procedures included fluoroscopy procedures, fluoroscopy with endoscopic guidance, and through-the-scope stent placements in a total of 606 patients. Stents were deployed in 97% of cases and symptoms were relieved in 87%, with almost half of patients achieving a full diet. In those who did not improve after stenting, failure was ascribed to disease progression in 61%, early stent migration in 20%, and poor stent placement/deployment in 15%. Severe complications such as perforation or bleeding occurred in 1.2%. However, minor complications occurred in almost 27% of patients, including stent obstruction or migration in 22%. Stent obstruction can be related to tumor ingrowth, overgrowth, food impaction, stent fracture or collapse, or prolapse of adjacent tissue. Both stent migration and obstruction can generally managed by re-stenting. Data on efficacy of palliative re-stenting are scant, but the practice seems reasonable, if anatomic factors permit, since this patient group as a whole may have even more advanced disease than primary stent candidates, and thus might inspire less enthusiasm for surgical bypass. Few studies have compared the outcomes of endoscopic stenting with those of surgical bypass. One retrospective study from Boston, of 27 patients with pancreatic cancer and gastric outlet obstruction demonstrated equivalent survival after surgical versus endoscopic palliation, but the endoscopic group had fewer re-hospitalizations and lower costs (68). Another study from Burlington, MA, albeit of only 23 patients, suggested that, in contrast to endoscopically stented patients, surgically bypassed patients had frequent postoperative delayed gastric emptying, longer postprocedural hospitalization, higher 30-day mortality, and shorter overall survival (69). A Swedish study of 36 patients demonstrated that stented patients had shorter hospitalizations, better 30-day survival, and better 30-day solid food tolerance than surgically bypassed patients (70). One Japanese study of 39 patients with malignant gastroduodenal obstruction suggested that surgery and endoscopic stenting had equivalent technical and clinical successes, survival, and complications, but

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that endoscopic stenting led to much faster resumption of oral intake (1 versus 9 days) and more frequent improvements in performance status (71). The largest nonrandomized study was done in Italy and compared 24 SEMS-treated patients with 23 open-surgical bypass patients (72). That study also showed the SEMS group to have less morbidity and shorter hospitalizations, as well as improved clinical success, 30-day survival, and overall survival. Indeed one small prospective, randomized trial of open gastrojejunostomy versus endoscopic stenting has been done in patients with malignant antropyloric strictures (73). With nine patients in each arm, there were equivalent 3-month outcomes, but shorter mean time to oral intake and shorter hospitalizations in the endoscopic group. In general, biliary stenting should be done before or at the time of gastroduodenal stenting for two reasons. First, duodenal stent insertion may lead to secondary biliary obstruction in some patients (65, 74, 75). Second, biliary stenting through an enteric stent can be technically challenging, especially if the stent is covering the major ampulla, which is commonly the case. In fact, there has been one study where nearly half of patients who had enteral SEMS placed required biliary interventions (76). Combined stenting of both the bile duct and the duodenum, in the same session, is successful in 91–94% of cases (61, 77). Thereby, in any patient without biliary obstruction who undergoes palliative stenting for malignant duodenal obstruction, biliary stenting should be attempted first. Furthermore, a biliary SEMS should be placed, to minimize the potential need for exchanging a plastic biliary stent, which has higher odds of obstructing sooner. In the patient in whom an enteral stent already exists, and biliary obstruction ensues, percutaneous biliary drainage may be the easiest option. That being said, it is possible to manage this situation endoscopically. One group described successfully placing biliary stents through metallic duodenal stents in 17 of 18 patients (78). Obviously these procedures are usually relegated to the most expert biliary endoscopists. Although endoscopic stenting often provides successful palliation of malignant gastric outlet obstruction, it does not have the durability of surgical bypass and does not address the gastroparesis that often accompanies advanced pancreatic cancer (77). Recent advances in endoscopic techniques have allowed for endoscopic facilitation of gastrojejunostomy without the need for an abdominal incision. These procedures require techniques to create apposition of the stomach and a loop of jejunum, as well as their fixation and creation of a fistula. Early techniques included the endoscopic placement of magnets to create apposition, as well as the use of endoscopic ultrasound to direct a needle and traction device to pull a loop of jejunum to the stomach (80–83). Once apposition is achieved a tract can be dilated, and different endoscopic suturing devices employed. Finally, there will be a subset of obstructed patients, usually with highly advanced disease, who cannot be properly bypassed or stented, or whose symptoms do not respond to those maneuvers. Such patients have persistent nausea and vomiting with an exceedingly poor quality of life. For these individuals, consideration should be given to placement of a venting gastrostomy, performed endoscopically or otherwise, which usually prevents persistent nausea and vomiting and often allows for some modestly restored oral intake (84).

470

N. Schmulewitz

4 Splanchnic Neurolysis Over 80% of patients with pancreatic cancer present with or develop pain (85–87). As most patients are not candidates for curative therapy, and the median survival is approximately 6 months (84), pain management is of paramount importance. Furthermore, patients with better pain management may have improved survival (86, 88). Unfortunately, in spite of its prevalence, pain in pancreatic cancer is not well understood, and multiple mechanisms may be at work. Potential causes include pancreatic ductal obstruction, inflammation, increased interstitial pressure, ischemia, and direct nerve infiltration or stretching by primary tumor or adenopathy (89–94). Additionally, peritumoral inflammation may sensitize nerves to further stimulation (89, 95, 96). As efficacy of therapies directed at these specific mechanisms has not been demonstrated, the usual standard of care for managing pain in unresectable pancreatic cancer involves escalating doses of narcotic analgesics. Unfortunately, narcotics can also impair quality of life when they cause mental status changes, unsteady gait, nausea, constipation, or make it unsafe for the patient to drive a car. Another means of palliating pain in advanced pancreatic cancer is splanchnic neurolysis. Sympathetic innervation of the pancreas, which includes fibers that mediate pain, arise from the tractus intermediolateralis in the spinal cord and travel along thoracic nerve roots 5 through 11. Afferent fibers from the pancreas, as well as other upper-abdominal viscera, directly enter the celiac plexus and then ascend along splanchnic nerves to the T5-T12 dorsal root ganglia. The celiac plexus itself is composed of a right and left ganglion that are wrapped around the aorta at the level of the celiac trunk, with a mean size of 3.1 × 3.2 × 3.5 centimeters, often lying between the celiac trunk and superior mesenteric artery (97), although the celiac trunk is the most reliable landmark (98). Pain fibers passing through the celiac plexus can be ablated chemically or transected with surgical splanchnicectomy. A placebo-controlled trial of intraoperative, chemical splanchnicectomy with 20 ccs of 50% alcohol versus saline in 137 patients with unresectable pancreatic cancer showed that patients with pre-existing pain had: (1) significant diminution of pain for a mean of over 3 months, (2) a 70% reduction in need for narcotics (versus 0% in placebo group), and (3) a prolonged survival (88). Furthermore, patients in that study without significant preoperative pain demonstrated a delay in onset of significant pain from 3 months to 7.2 months. A less invasive surgical technique to transect afferent splanchnic innervation is thoracoscopic splanchnicectomy, which can be done unilaterally or bilaterally, with >80% of patients achieving clinical benefit of pain reduction (99–103). Interestingly, there is no clear evidence that bilateral splanchnicectomy imparts an advantage over unilateral splanchnicectomy, particularly as an initial intervention, and so a left-sided unilateral technique is common. Nevertheless, nonoperative chemical splanchnicectomy tends to be the preferred approach in unresectable patients. Percutaneous celiac plexus blocks have been done for >90 years. The most common percutaneous route is a posterior one, where a 15- to 20-cm needle is passed from a position posterolateral to the first lumbar vertebra alongside the vertebral body so that the tip reaches a position just anterior

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to the spine. There are different posterior approaches, including retrocrural, transcrural, and transaortic ones, and these are accomplished under fluoroscopic or CT guidance. Anterior approaches have also been described, usually with ultrasound or CT guidance, although bowel and solid organ puncture are concerning risks. Although the celiac ganglia are not visible by fluoroscopy or CT, various approaches are effective because liquids spread around the preaortic space when injected nearby within the retroperitoneum, even if injected retrocrurally (104, 105). For patients with cancer pain, celiac plexus neurolysis (CPN) is accomplished by injecting phenol, or, more commonly, alcohol (usually 20 ccs of 98% alcohol), after first injecting bupivacaine, a local anesthetic, to avoid the pain associated with alcohol administration to neural tissue. The traditional technique involved injection of alcohol on the day following an “analgesic test dose” of bupivacaine. More contemporary practice allows for the alcohol injection to immediately follow the local anesthetic test dose and the patient’s elicited response to the latter. A review of 24 studies of percutaneous CPN, in which 63% of patients had pancreatic cancer, demonstrated partial to complete pain relief in about 90% of patients at 3 months, and 70–90% of patients at the time of death (106). However, patient selection may be important. Some data suggest that factors predictive for CPN success are tumors in the head of the pancreas, shorter duration of cancer pain, and tumors undergoing chemotherapy and/or radiation (107–109). These factors are intuitive because they relate to a lower likelihood of peripancreatic nerve involvements. More specifically, smaller tumors that present earlier, as do lesions in the head that cause jaundice and tumors in general that have been symptomatic for shorter periods tend to have less peripancreatic nervous tissue invasion. Similarly, patients receiving local or systemic therapy may have their tumors shrink away from peripancreatic nerves. Contrarily, large tumors in the body and/or tail may impede the ability of injected liquid to freely “bathe” the area around the celiac ganglia and may involve peripancreatic nerves that do not respond to celiac neurolysis. That being said, CPN later in the disease course still can often be effective (110). The most common side effects of CPN include pain (up to 96%), hypotension (up to 38%), and diarrhea (up to 44%) (109). The pain results from alcohol’s immediate caustic effects on nerves and lasts for 1–2 days. Hypotension occurs transiently after the procedure and presumably reflects splanchnic vasodilation resulting from acute splanchnic neuronal injury, but the mechanism is not understood. Significant hypotension can often be averted by prophylactic volume expansion during and immediately after the procedure. Diarrhea, when it occurs, usually only lasts a couple of days, and not more than a week. Rare CPN complications include vascular injury, retroperitoneal infection, pneumothorax, chylothorax, chest pain, hematuria, hiccupping, and gastrointestinal motility disruptions such as chronic diarrhea or persistent gastroparesis (106, 111–113). However, the most important potential complication is neurologic injury. Neurologic injuries are the most concerning potential complications of percutaneous CPN, regardless of approach. These injuries include lowerextremity weakness, paresthesias, or paraplegia, the latter occurring in up to 1% of patients, mediated by direct injury to the cord or somatic nerves or cord ischemia from needle injury to the artery of Adamkiewicz (106, 114–117).

472

N. Schmulewitz

CPN practice has changed a bit with the availability of the endoscopic approach, done via EUS. First reported in the mid 1990s, EUS-CPN is now practiced in most centers where EUS is available. Patients are sedated for endoscopy and a linear echoendoscope is passed near the esophagogastric junction, where posterior-wise rotation of the ultrasound probe allows for visualization of the aorta. As the probe is advanced distally, the diaphragm disappears from between the esophageal wall and aorta and the origins of the celiac trunk and superior mesenteric artery below it will come into view. A hollow-gauge needle (19G, 22 G, or the newly available 20 G CPN needle with side holes for expeditious injections) is passed through the posterior gastric wall and the needle tip placed anterior and superior to the celiac trunk takeoff. Here 4–6 ccs of 0.25–0.50% bupivacaine is injected, followed by 20 ccs of 98% alcohol. Some operators choose a single midline injection, whereas others prefer two divided doses injected just laterally to the right and left of the celiac trunk. In one study of EUS-CPN in 30 patients, patients demonstrated a 79– 88% reduction in pain scores and a 49% reduction in pain medication usage at 12 weeks (118). Another study of 58 patients yielded reduction in pain in 78% of patients but without significant change in narcotic usage (109). The biggest advantage of EUS-CPN is the close proximity of the ultrasound probe in the stomach to the target area adjacent to the celiac trunk, which is usually 50 Gy was 19.2% and 31% for IMRT and 3DCRT, respectively. The median volume of small bowel exceeding 60 Gy was 12.5% and 19.8% for IMRT and 3DCRT, respectively. The normal-tissue complication probability model predicted a small bowel complication probability of 9.3% with IMRT compared to 24.4% with 3DCRT delivery of dose (p = 0.021). There is very little clinical experience with the use of IMRT in pancreatic cancer. The Wayne State University group has reported on 15 patients (seven treated adjuvantly after curative resection and eight treated for unresectable disease) (44). IMRT was planed using the CORVUS system and delivered with a segmented multileaf collimator, using a 6 MV photon beam and ten intensity steps. Two target volumes were entered; target 1 consisted of the gross tumor volume (in unresectable cases) or the tumor bed (in post surgical cases) and target 2 consisted of the draining lymph nodes. Both targets were treated simultaneously in 25 daily fractions, 5 days a week. In the postoperative setting the total dose to target 1 was 45–54 Gy (median: 54 Gy). For unresectable disease the dose was 54–55 Gy (median: 54 Gy). The total dose to target 2 was 45 Gy in all patients. Patients were treated with one of two six-field beam arrangements that had been found to produce superior dose distributions in prior studies. Capecitabine was given at 1,600 mg/m2/ day in two divided doses, 5 days per week, concurrently with radiotherapy. In addition, most patients (73%) received gemcitabine-based chemotherapy. Systemic chemotherapy was given before, after, or both before and after chemo-radiotherapy in 47%, 7%, and 20% of patients, respectively. Treatment was tolerated well. Only one patient (7%) developed grade III toxicity: gastric ulceration that responded to medical management. Encouragingly, in the unresectable group, the 1-year actuarial survival rate was 69% and two patients (25%) converted to resectability. At the University of Michigan, we have explored the potential of IMRT to escalate the dose to the target while respecting normal tissue tolerances. We hypothesized that by allowing greater heterogeneity across the PTV, we will be able to deposit substantially greater doses within the PTV. To drive the process of dose escalation in a biologically meaningful way, we elected to use the concept of gEUD. Originally introduced by Niemierko (33), gEUD is the biologically equivalent dose that, if given uniformly, would lead to the same level of tumor cell kill as the actual nonuniform dose distribution at hand. Subsequently, the concept was extended to normal tissues (34). Additional work showed that gEUD can be used for IMRT optimization (45). Comparing dose-volume–based and gEUD–based optimization of IMRT plans for prostate and head and neck cancer

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patients, Wu et al. (45) found that the latter allowed similar or better target coverage with a parallel reduction in dose to OAR. We compared 3DCRT and IMRT treatment plans for 15 patients with locally advanced unresectable pancreatic cancer. GTV was defined as the imaged tumor on a pancreas protocol CT. PTV was defined as the GTV plus 1 cm expansion, as per planning guidelines in our previous institutional clinical trials. In addition, within the GTV, we defined the area of vascular involvement precluding surgical resection as the surgical boost volume (SBV). One of the level 2 goals (see the following) was to preferentially escalate the dose within this volume. The constraints for OAR in the IMRT plan are detailed in Table 28.1. A previously described (44), six-field non-coplanar beam arrangement with 5 mm IMRT beamlets was used (Fig. 28.4A).

Fig. 28.4 Representative IMRT plan in a patient with unresectable pancreatic cancer. A. The six-field non-coplanar beam arrangement typically used, with their intensity maps. Also shown are the PTV (pink), duodenum (yellow), small intestine (tan), stomach (green), kidneys (blue), and liver (brown). B. The GTV (pink) and surgical boost volume (red). C. Dose distribution and OAR and PTV. Note the high dose achievable in a large part of the PTV and the rapid falloff, meeting stringent constraints in OAR. D. Dose-volume histograms comparing IMRT (solid lines) and 3DCRT (dashed lines). Note that IMRT results in substantial increases in dose to PTV and substantial decreases in dose to OAR

492

A.C. Spalding, E. Ben-Josef

Optimization was performed using lexicographic ordering. The hierarchical planning goals are categorized into several levels of priority. The various priority level goals are optimized sequentially, one level of priority at a time, with the least important planning goals addressed last. By subdividing a large multicriteria problem into several somewhat smaller problems based on priority levels, the complex space of tradeoffs can be significantly simplified, greatly decreasing the need for iterative optimization trials before a solution that satisfies the goals of the protocol is found. In this study, the OAR dose goals were identified as the most important, therefore addressed at the first level. After the dose limits for the OARs were ensured, the next goal of escalating the PTV dose (maximize the minimum target dose, maximize the target gEUD, and keep maximum target dose